Wax synthesis in Brassica oleracea as modified by trichloroacetic acid and glossy mutations

Two different mutations in Brassica oleracea, gl₅ and gl₄ have been re-investigated using acetate-1-¹⁴C labelling in an attempt to define more closely the nature of the genetic blocks to wax synthesis. It has been found that gl₅ is a mutation which blocks elongation in the Step C₂₈–C₃₀. The mutation gl₄ exhibits no elongation block and could be blocked in the decarboxylation Step C₃₀–C₂₉. 0·1 mM TCA supplied in the culture solution of cauliflower seedlings affected the leaf surface by producing a glossy appearance similar to that induced by gl₃ and gl₄. At this concentration growth was not inhibited and the appearance of the plants was normal except for the surface wax. The amount of surface wax produced was about 40% of that in untreated seedlings on a leaf area basis. Slight, but significant changes in wax composition were noted, mainly involving a reduction in C₃₀ acids and aldehydes, a slight reduction (33–29%) in alkane content, and a marked difference in chain length composition of the alkanes with C₂₇ increased relative to C₂₉. Over a range of concentrations from 0·1–1 mM, TCA inhibited incorporation of label from acetate-1-¹⁴C into C₃₀ acids and aldehydes more than into C₂₈ at concentrations 0·4–0·8 mM while label tended to accumulate in C₂₄ and C₂₆ acids; thus elongation C₂₈–C₃₀ was especially sensitive to TCA. TCA also inhibited incorporation into primary alcohols and esters almost as much as into C₂₉ compounds. In spite of relatively specific effects on incorporation of label into longer chain lengths, the resulting block to C₃₀ synthesis is not sufficient to make much difference to the overall rate of C₂₉ synthesis. Both results of analysis of wax from whole plants and experiments with tissue slices in vitro indicated that the effect of TCA in reducing the glaucousness of the leaf surface is a combination of overall reduction of wax synthesis together with slight but significant changes in wax composition.     

Epicuticular waxes of Secale cereale and Triticale hexaploide leaves

Wax on leaves of rye and of hexaploid Triticale (60–70-day-old plants) contains hydrocarbons (6–8%), esters (10%), free alcohols (14-8%), free acids (3%), hentriacontane-14,16-dione (39–45%), 25 (S)-hydroxyhentriacontane-14,16-dione (13–11%) and unidentified (14–15%). Diesters (1–3%) are also present in rye wax. Compositions of hydrocarbons (C₂₇-C₃₃) and esters (C₂₈,C₅₈) are similar for both waxes. Free and combined alcohols of rye wax are mainly hexacosanol but alcohols of Triticale wax are mainly octacosanol. The composition of Triticale wax is close to that of its wheat parent Triticum durum (cv. Stewart 63). Esters of wax from ripe rye contain 58% of trans 2,3-unsaturated esters. *NRCC No. 14033.     

Epicuticular waxes of four eragrostoid grasses

The major components of Sporobolus airoides wax were hydrocarbons (37%, C₂₇–C₃₃), those of Bouteloua curtipendula and Eragrostis trichoides waxes esters (28% and 31%, respectively) and those of Muhlenbergia wrightii wax free alcohols (57%, almost entirely C₂₈). Free alcohols formed 22% of the wax from B. curtipendula, 19 % of the wax from E. trichoides and 10% of the wax from S. airoides; the compositions ranged from C₂₆ to C₃₂ with C₃₂ the major component. These alcohol compositions are similar to those found for other species in the subfamily Eragrostoideae. The esters contain 32–46% of acylated triterpenols, principally α- and β-amyrins. Aldehydes were present in all the waxes except for that from S. airoides.     

Alkyl esters from pinus radiata foliage epicuticular wax

Wax esters from the epicuticular wax of juvenile and mature-tree Pinus radiata foliage have been shown by capillary column GC-MS to consist mainly of short chain (C₆–C₁₂)alkanols esterified with long chain acids (C₂₄–C₃₂) and long chain alkanols (C₂₄–C₃₂) esterified with short chain acids (C₆-C₁₄) in a non-random manner. Mature-tree foliage wax esters also contained nonacosan-10-ol esterified with dodecanoic and tetradecanoic acids.     

Absence of long chain aldehydes in the wax of the Glossy II mutant of maize

Wax from the glll mutant of maize lacks aldehydes, which constitute 20 % in the normal genotype. The absence of aldehydes is not associated with a block in the synthesis of alcohols. Moreover in contrast to the wild type, glll wax is characterized by a higher content of C₁₆ and C₁₈ free acids, with a clear defect in the synthesis of C₂₄, C₂₆ and C₂₈ homologues. The results from this study are taken as evidence that the wild type elongation-decarboxylation I (EDI) pathway, leading to the synthesis of all the wax classes of compounds except esters, may be split into an early (EDIa) and a late (EDIb) group of reactions. Mutant glll is apparently defective at the EDIa, governing the synthesis of C₂₄–C₂₈ fatty acyl chains.     

Epicuticular waxes from Agropyron dasystachyum,agropyron riparium and Agropyron elongatum

Epicuticular waxes from whole plants of Agropyron dasystachyum var. psammophylum, A. riparium and A. elongatum contain hydrocarbons (5–8 %), long chain esters (12–15%) and free acids (2–5%). The major esters are C₃₄C₅₆ esters derived from C₁₆C₃₀ acids and alcohols (1-hexacosanol is the major alcohol) but C₃₁, C₃₃ and C₃₅ esters (3–11%) are also present. The latter esters are C₁₈ and C₂₀ acid esters of C₁₃ and C₁₅ 2-alkanols. A. dasystachyum wax contains 2% free alcohols, that of A. riparium contains 17% and that of A. elongatum 11% (1-hexacosanol is the major alcohol in each). Diesters (2%), C₈C₁₂ diols esterified by (E)-2-alkenoic acids, are present in A. riparium wax. Hentriacontane-14,16-dione is present: 29% in A. dasystachyum wax and 32% in A. riparium wax, but only 5% in A. elongatum wax. 25-Oxohentriacontane-14,16-dione forms 14% of A. dasystachyum wax and 27% of A. elongatum wax but the oxo β-diketones of A. riparium wax (5%) consist of both 10-oxo- and 25-oxohentriacontane-14,16-diones in the ratio 4:1. Hydroxy β-diketones of the waxes are 25- and 26-hydroxyhentriacontane-14,16-diones; in A. dasystachyum (20%) the ratio is 3:1, in A. elongatum (20%) the ratio is 9:1 but in A. riparium (5%) it is ca 1:2. The configuration of the hydroxyl group in the 26-hydroxy β-diketone is opposite to that in the 25-hydroxy derivative. The unusual composition of the oxygenated β-diketones of A. riparium confirms that this species should be regarded as separate from A. dasystachyum. Wax from A. elongatum also contains 4-hydroxy-25-oxohentriacontane-14,16-dione (4%) and an unusual oxo-β-ketol, 18-hydroxy-7,16-hentriacontanedione (2%), both these components are probably derived biosynthetically from the 25-oxo β-diketone which is the major component of this wax. Syntheses of racemic 18-hydroxy-7,16-hentriacontanedione and of a model β-ketol, 12-hydroxy-10-pentacosanone, are described.     

Epicuticular waxes of Sorghum and some compositional changes with plant age

Epicuticular wax from mature plants of Sorghum bicolor SD-102 was compared with that from panicles and seedlings of the same variety at the fourth-fifth leaf stage of growth. The composition of wax from SD-102 panicles was quite different from that of mature leaf blades and sheaths. Free fatty alcohols were the dominant class of wax from SD-102 seedlings and C₃₂ was the major homologue of alcohols and aldehydes. For comparison purposes, the epicuticular waxes from whole plants of two other S. bicolor varieties, Alliance A and Martin A, grown up to the tasseling stage, have been analysed. The major wax components were free fatty acids. The typical chain lengths of aldehydes, free alcohols and free fatty acids were C₂₈ and C_30.p-Hydroxybenzaldehyde was not a wax component of the studied varieties of sorghum.     

Isoflavone,wax and triterpene constituents of Wyethia mollis

The hexane extract of Wyethia mollis contains the n-alkanes C₁₅-C₁₈, C₂₀-C₂₅, C₂₇ and C₂₉. Linoleic acid was the only detectable acidic component. A mass spectral analysis of the wax ester fraction indicated that it was a mixture of homologues, the saturated even-carbon acids n-C₁₆-C₃₀ esterfield with the saturated even-carbon alcohols n-C₁₈-C₂₆. The chloroform extract yielded the known isoflavones santal and 3′-O-methylorobol along with a new lanostane-type triterpene, 22,25-epoxy-lanosta-7:9(11)-dien-3-one. The wide distribution of n-alkanes and the decreased odd-even carbon ratio are consistent with the proposed primitive nature of this plant.     

Intraspecific Variation in the Epicuticular Wax Composition of Four Species of Chionochloa

Intraspecific variation in four New Zealand species of Chionochloa, C. flavescens, C. pallens, C. rigida; and C. rubra, was investigated by examining the major carbon chain lengths of fatty acids, alcohols, aldehydes, wax esters and alkanes of the epicuticular waxes. The major even-carbon chain lengths ranged generally from C₂₄ to C₃₂ in the acids, alcohols and aldehydes; C₂₉ to C₃₃ in the alkanes; and even-carbon chains between C₃₆ and C₅₂ in the wax esters. A computer program was used to calculate the degree of similarity between samples in terms of chain length distribution. In C. rigida eastern and western South Island localities were identified; in C. flavescens Canterbury and Nelson, western South Island and southern North Island regions were recognized; and C. pallens and C. rubra were divisible into four regions; Canterbury, Nelson, western South Island and southern North Island. The possible elongation-decarboxylation pathways and the specificity of the enzymes in the biosynthetic pathways of epicuticular wax synthesis suggest the possibility that the northwest Nelson region could be a biogenetic centre from which wax synthesis has diversified along three routes, one to the western South Island, another to eastern South Island and the third to southern North Island. Identification of each of the four species based on the distribution of the carbon chain lengths in the individual lipid fractions is impossible unless the locality of collection is known. Intraspecific variation in lipid composition is not coincident with patterns of variation already reported.     

Epicuticular wax of Poa ampla leaves

Leaf wax of a glaucous variety of Poa ampla contains hydrocarbons (5%, C₂₃–C₃₅), esters (9%, C₃₆–C₅₆), free acids (3%, C₁₆–C₃₄), free alcohols (6%, mainly C₂₆); hentriacontane-14,16-dione (14%), 5-oxohentriacontane-14,16-dione (1%); hydroxy β-diketones (56%) and unidentified material (6%). The hydroxy β-diketones, which are more abundant in this wax than in others, were shown by ¹³C NMR to consist of 4-hydroxy (15%), 5-hydroxy (70%) and 6-hydroxy (15%) hentriacontane-14,16-diones.     

Isolation of n-paraffins and sec-alcohols from Isotachis japonica

Two homologous series of n-paraffins and sec-alcohols from C₂₀ to C₃₄ or C₃₅ were isolated a wax constituents of a liverwort, Isotachis japonica. In both series, odd-members were predominant compared with even-members: the ratio of the total amount of odd- to total amounts of even-members was 3·9 in the n-paraffin homologues, and 16·5 in the sec-alcohol homologues.     

Occurrence and Carbon Metabolism of Green Nonsulfur-like Bacteria in Californian and Nevada Hot Spring Microbial mats as Revealed by Wax Ester Lipid Analysis

Green nonsulfur-like bacteria (GNSLB) in Yellowstone hot spring microbial mats have been extensively studied and are thought to operate both as photoheterotrophs and photoautotrophs. Here we studied the occurrence and carbon metabolisms of GNSLB by analyzing the distribution and isotopic composition of their characteristic wax ester lipids in four Californian and Nevada hot spring microbial mats at a range of temperatures (37–96°C). The distribution of wax esters varied strongly with temperature. At temperatures between 50–60°C the wax ester composition in each of the four hot spring microbial mats was dominated by C₃₀ to C₃₆ wax esters, consisting of mixtures of C₁₅-C₁₈ n-alkyl and branched fatty acids and alcohols, typical for GNSLB. Stable carbon isotopic analysis showed that these wax esters were only depleted by 5 to 10‰ compared to dissolved inorganic carbon in the overlying water, suggesting that these GNSLB were mainly autotrophic. However, analysis of different depth layers of one microbial mat showed that these GNSLB wax esters were increasingly depleted in ¹³C with depth, suggesting that photoautotrophy mainly occurred in the top layer of the mat. ¹³C-depleted C₃₆-C₄₄ wax esters were found in one hot spring at high temperatures (77–96°C) and are likely derived from allochtonous plant waxes. At several lower temperature sites (35–40°C) the wax esters were predominantly composed of C₂₈, C₃₀ and C₃₂ wax esters consisting of mixtures of C₁₄-C₁₆ fatty acids and n-alkanols and were depleted in ¹³C by 15–20‰ relative to dissolved inorganic carbon, suggesting they may be derived from heterotrophic organisms. Our results indicate that autotrophic GNSLB occur widely in hot springs and that diverse groups of organisms contribute to the pool of wax ester lipids in hot spring environments.     

Epicuticular wax of Pinus radiata needles

Epicuticular wax isolated from the cotyledons and primary needles of 10-week-old Pinus radiata seedlings is similar in composition and contains 86% neutral compounds, viz. alkyl esters (25%, C₂₄–C₆₄), nonacosan-10-ol (52%), heptacosane-5,10-diol (2%), nonacosane-4,10-diol, nonacosane-5,10-diol, and nonacosane-10,13-diol (total 12%) and estolides, MW ca 800 (2%), MW ca 1100 (6%), and MW ca 1500 (1%). The acidic fraction (14%) contains n-acids (78%, C₁₂–C₃₂) and diterpene acids (22%, mainly abieta-8,11,13-trien-18-oic, with lesser amounts of pimara-8(14),15-dien-18-oic, isopimara-7,15-dien-18-oic and hydroxylated aromatic, diene and mono-ene acids). Wax isolated from primary needles of 1-yr-old seedlings had a similar neutral fraction composition, but the acidic fraction contained predominantly the diterpene acid mixture, with only trace amounts of n-acids. The wax from 1-yr-old secondary, needles from P. radiata forest trees aged 5 yr and 40 yr contained an acid fraction (12% 5 yr, 17% 40 yr trees) comprising the diterpene acid mixture, with trace amounts of n-acids together with ω-hydroxy acids (C₁₂, C₁₄ and C₁₆). The neutral fraction from both young and old trees had a similar composition containing alkyl esters (7%, C₂₄–C₆₆), estolides (90%, MW 566-ca 1500), nonacosan-10-ol (2%) and the heptacosane and nonacosane diols (1%). During growth and maturation of P. radiata, the nonacosan-10-ol content of the needle wax decreases while the proportion of estolides and diterpene acids increases, the latter probably being located around the stomatal pore.     

Compositae Plants Differed in Leaf Cuticular Waxes between High and Low Altitudes

We sampled eight Compositae species at high altitude (3482 m) and seven species at low altitude (220 m), analyzed the chemical compositions and contents of leaf cuticular wax, and calculated the values of average chain length (ACL), carbon preference index (CPI), dispersion (d), dispersion/weighted mean chain length (d/N), and C₃₁/(C₃₁ + C₂₉) (Norm31). The amounts of total wax and compositions were significantly higher at high altitude than at low altitude, except for primary alcohol, secondary alcohol, and ketone. The main n‐alkanes in most samples were C₃₁, C₂₉, and C₃₃. Low altitude had more C₃₁ and C₃₃, whereas more C₂₉ occurred at high altitude. The ACL, CPI, d, d/N, and Norma 31 were higher at low altitude than at high altitude. The fatty acid and primary alcohol at low altitude contained more C₂₆ homologous than at high altitude. More short‐chain primary alcohols were observed at high altitude. At low altitude, the primary alcohol gave on average the largest amount, while it was n‐alkane at high altitude. These results indicated that the variations of leaf cuticular waxes benefited Compositae plants to adapt to various environmental stresses and enlarge their distribution.     

Very-Long-Chain Wax Constituents from Primula veris and P. acaulis: Does the Paradigm of Non-Branched vs. Branched Chain Dominance Universally Hold in all Plant Taxa?

Herein n-, iso- and anteiso-series of very-long-chained (VLC) alkanes (C₂₁–C₃₅), fatty acid benzyl esters (FABEs; C₂₀–C₃₂), and 2-alkanones (C₂₃–C₃₅) were identified in the wax of Primula veris L. and P. acaulis (L.) L. (Primulaceae). For the very first time in a sample of natural origin, the presence of iso- and anteiso-VLC FABEs and 2-alkanones was unequivocally confirmed by synthetic work, derivatization, and NMR. It should be noted that the studied species produced unusually high amounts of branched wax constituents (e. g., >50 % of 2-alkanones were branched isomers). The domination of iso-isomers, probably biosynthesized from leucine-derived starters, is a unique feature in the Plant Kingdom. The plant organ distribution of these VLC compounds in P. acaulis samples (different habitats and phenological phases) pointed to their possible ecological value. This was supported by a eutectic behavior of binary blends of FABEs and alkanes, as well as by high UV-C absorption by FABEs.     

The composition of external lipids from adult whiteflies,Bemisia tabaci and Trialeurodes vaporariorum

The total surface lipids, including the wax particles, of the adult whiteflies of Bemisia tabaci and Trialeurodes vaporariorum were characterized. At eclosion, there were similar amounts of long-chain hydrocarbons, aldehydes, alcohols and wax esters. Within a few hours post-eclosion, long-chain aldehydes and long-chain alcohols were the dominant surface lipid components, C₃₄ on B. tabaci and C₃₂ on T. vaporariorum. Hydrocarbons, mainly n-alkanes, were minor components of the surface lipids. The major wax esters were C₄₆ on B. tabaci and C₄₂ on T. vaporariorum. The major acid and alcohol moieties in the wax esters of B. tabaci were C₂₀ and C₂₆, respectively, and of T. vaporariorum were C₂₀ and C₂₂, respectively. Both B. tabaci and T. vaporariorum had a minor wax ester composed of the fatty acid C_18:1 esterified to the major alcohols, C₃₄ and C₃₂, respectively. Bemisia were readily distinguished from Trialeurodes based on the composition of their wax particles and/or their wax esters; however, no differentiating surface lipid components were detected between biotypes A and B of B. tabaci.     

Epidermal structures and composition of epicuticular waxes of Sedum album sensu lato (Crassulaceae) in Balkan Peninsula

Sedum album is a succulent plant, with phenotypic variability resulting in delimitation of a number of taxa with diverse taxonomic recognition. We analyzed the taxa from Sedum album in broad sense (S. album, S. micranthum, S. athoum and S. serpentini) from Balkan Peninsula, to check whether the variability of their epidermal structures and epicuticular wax composition corresponds to their taxonomic reliability. Epidermal structures and epicuticular waxes were analyzed using light and scanning electron microscopy, gas chromatography-mass spectrometry and gas chromatography-flame ionization detector chromatography and multivariate statistical techniques. Analyses of studied taxa have shown that significant systematic characters include adaxial epidermal and guard cell length, stomatal frequency and subsidiary cells area on abaxial epidermis. The content of n-alkanes C₂₉, C₃₀, C₂₇, C₃₂ and C₃₃ was shown to be valuable criterion for their phytochemical delimitation. The comparison of epicuticular wax microstructures has shown no significant differences. Results indicate presence of two (micromorphology) or three (phytochemistry) well-defined groups of populations, with S. serpentini manifesting a considerable level of separation toward the others. Sedum athoum have shown weak to moderate degree of differentiation, while S. micranthum has shown general resemblance to S. album.     

Variations of the Composition of the Leaf Cuticular Wax among Chinese Populations of Plantago major

Plantago major L. grows in a very wide range of regions in China and exhibits great variations among populations. The analysis of the cuticular‐wax composition provides a potential approach to classify populations of P. major confronting different environmental conditions. Twelve populations of P. major and five populations of P. depressa Willd ., distributed over regions with average annual temperatures ranging from ?2.0 to 18.4°, were sampled, the variation of the composition of their cuticular waxes was analyzed, and their values of average chain length (ACL) and carbon preference index (CPI) were calculated. Great intra‐ and interspecies variations were observed for the total wax contents. The average annual temperature of the habitats was significantly correlated with the relative contents of the dominant n‐alkanes with an odd number of C‐atoms, but not with the wax contents. With an increasing average annual temperature, the relative contents of n‐alkanes C₂₉ and C₃₁ decreased, whereas those of C₃₃ and C₃₅ as well as the values of ACL_total and ACL_27–33 increased. Cluster analysis based on the pattern of the n‐alkane distribution allowed to clearly separate the populations of P. major according to the average annual temperature of their habitats, but not to separate the populations of the two species. Hence, the pattern of the n‐alkane distribution might be a good taxonomic marker for P. major at the intraspecies level, but not at the interspecies level. Nevertheless, a small difference between the populations of the two species was observed concerning the values of ACL_total and CPI_total, implying the potential use of these indices for the classification of the populations of the two species at the interspecies level.     

Wax composition of sargassum fulvellum

Sixty-seven compounds were characterized in the wax of Sargassum fulvellum. Characteristic components were the 5-methylhexyl esters of octanoic, decanoic, lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic, and the 2-ethylhexyl esters of the same acids. The wax of S. fulvellum contains hydrocarbons (1.6%), esters (21.8%), free acids (74.9%) and free alcohols (0.3%). The principal free alcohols range in chain length only from C₆ to C₇.     

Intracuticular lipids of spinach leaves

The cuticular wax and cutin components of the cuticular membranes isolated from the leaves of two spinach cultivars have been determined. The membranes contain about 0·007 mg/cm² of cuticular wax which comprises monobasic acids (C₁₆–C₃₈) with hexadecanoic as the major component. The amounts of cutin are comparable with those of cuticular wax and the monomeric constituents are predominantly C₁₈ epoxy compounds. The most abundant monomer is 9,10-epoxy-18-hydroxyoctadecanoic acid (up to 63%) together with substantial amounts of 9,10,18-trihydroxyoctadecanoic acid (up to 22%). Also present are 9,10-epoxyoctadecane-1,18-dioic acid (6–7%) dihydroxyhexadecanoic acid (3–4%) and ω-hydroxymonobasic and fatty acid fractions. The tentative identification of two minor components, 18-hydroxyoxooctadecanoic and 9,10-epoxy-12,18-dihydroxyoctadecanoic acids, is also made. Although spinach membranes have a delicate structure their cutin composition is essentially similar to that of much more substantial membranes.