Homologous very-long-chain 1,3-alkanediols and 3-hydroxyaldehydes in leaf cuticular waxes of Ricinus communis L

Surface extracts from primary leaves of Castor bean were found to contain 1.8 microg cm(-2) of cuticular waxes. The mixture comprised alkanes (C(26)-C(29)), primary alcohols (C(22)-C(38)), aldehydes (C(26) and C(28)), fatty acids (C(20)-C(34)) and triterpenoids (lupeol, beta- and alpha-amyrin). Besides, a series of n-alkane-1,3-diols was detected, with chain lengths ranging from C(22) to C(28), a strong predominance of even-numbered homologs, and a maximum for hexacosane-1,3-diol. Seven other compounds were assigned to a novel class of wax constituents and identified as homologous unbranched 3-hydroxyaldehydes ranging from C(22) to C(28). As the chain length distribution of this series closely paralleled the homolog pattern of 1,3-diols, it seems likely that both compound classes are biosynthetically related.     

The positional sterile (ps) mutation affects cuticular transpiration and wax biosynthesis of tomato fruits

Cuticular waxes are known to play a pivotal role in limiting transpirational water loss across primary plant surfaces. The astomatous tomato fruit is an ideal model system that permits the functional characterization of intact cuticular membranes and therefore allows direct correlation of their permeance for water with their qualitative and quantitative composition. The recessive positional sterile (ps) mutation, which occurred spontaneously in tomato (Solanum lycopersicum L.), is characterized by floral organ fusion and positional sterility. Because of a striking phenotypical similarity with the lecer6 wax mutant of tomato, which is defective in very-long-chain fatty acid elongation, ps mutant fruits were analyzed for their cuticular wax and cutin composition. We also examined their cuticular permeance for water following the developmental course of fruit ripening. Wild type and ps mutant fruits showed considerable differences in their cuticular permeance for water, while exhibiting similar quantitative wax accumulation. The ps mutant fruits showed a five- to eightfold increase in water loss per unit time and surface area when compared to the corresponding wild type fruits. The cuticular waxes of ps mutant fruits were characterized by an almost complete absence of n-alkanes and aldehydes, with a concomitant increase in triterpenoids and sterol derivatives. We also noted the occurrence of alkyl esters not present in the wild type. Quantitative and qualitative cutin monomer composition remained largely unaffected. The significant differences in the cuticular wax composition of ps mutant fruits induced a distinct increase of cuticular water permeance. The fruit wax compositional phenotype indicates the ps mutation is responsible for effectively blocking the decarbonylation pathway of wax biosynthesis in epidermal cells of tomato fruits.     

Cuticular wax composition of Salix varieties in relation to biomass productivity

The leaf cuticular waxes of six Salix clones (one Salix miyabeana, one Salix dasyclados, one Salix eriocephala, two Salix purpurea, and one interspecific hybrid of Salix eriocephala x interior) with different biomass productivities were characterized by gas chromatography-mass spectrometry. Total wax content ranged from 6.3 to 16.8 microg cm(-2), and two distinct patterns of wax were measured. The wax from leaves of S. dasyclados 'SV1' differed from all other clones and was dominated by fatty acids (42%), high concentrations of n-alkanes (25%) and n-alcohols (28%), with low n-aldehyde content (4%). All other clones produced cuticular wax dominated by n-alcohols (32-51%), particularly 1-hexacosanol, with fatty acids (14-37%) and n-aldehydes (19-26%) present in lower abundances. Clones of Salix grown under identical environmental conditions produce noticeably different amounts of cuticular wax. In contrast to previous studies of Salix, total wax content was independent of biomass productivity, measured as basal area, suggesting that wax production is not directly linked with woody biomass production by shrub willows under these site conditions.     

Very long-chain phenylpropyl and phenylbutyl esters from Taxus baccata needle cuticular waxes

The cuticular wax of Taxus baccata L. needles was found to contain four different classes of long-chain esters that were identified by various chemical transformations with product assignment employing GC-MS. Homologous series of (1) 3-(4'-hydroxyphenyl)-propyl esters of C(20)-C(36) fatty acids, (2) 4-(4'-hydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids, (3) 3-(3',4'-dihydroxyphenyl)-propyl esters of C(20)-C(32) fatty acids, and (4) 4-(3',4'-dihydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids were identified. The four compound classes amounted to 0.1-3.6 micro g/cm(2) of needle surface area, corresponding to 0.2-7.6% of the wax mixture, respectively. While both phenylpropyl ester series had a maximum for the homolog containing tetracosanoic acid, in the phenylbutyl esters homologs containing eicosanoic and docosanoic acids predominated.     

Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces

The reaction of plants to environmental factors often varies with developmental stage. It was hypothesized, that also the cuticle, the outer surface layer of plants is modified during ontogenesis. Apple plantlets, cv. Golden Delicious, were grown under controlled conditions avoiding biotic and abiotic stress factors. The cuticular wax surface of adaxial apple leaves was analyzed for its chemical composition as well as for its micromorphology and hydrophobicity just after unfolding of leaves ending in the seventh leaf insertion. The outer surface of apple leaves was formed by a thin amorphous layer of epicuticular waxes. Epidermal cells of young leaves exhibited a distinctive curvature of the periclinal cell walls resulting in an undulated surface of the cuticle including pronounced lamellae, with the highest density at the centre of cells. As epidermal cells expanded during ontogenesis, the upper surface showed only minor surface sculpturing and a decrease in lamellae. With increasing leaf age the hydrophobicity of adaxial leaf side decreased significantly indicated by a decrease in contact angle. Extracted from plants, the amount of apolar cuticular wax per area unit ranged from only 0.9 microgcm(-2) for the oldest studied leaf to 1.5 microgcm(-2) for the youngest studied leaf. Differences in the total amount of cuticular waxes per leaf were not significant for older leaves. For young leaves, triterpenes (ursolic acid and oleanolic acid), esters and alcohols were the main wax components. During ontogenesis, the proportion of triterpenes in total mass of apolar waxes decreased from 32% (leaf 1) to 13% (leaf 7); absolute amounts decreased by more than 50%. The proportion of wax alcohols and esters, and alkanes to a lesser degree, increased with leaf age, whereas the proportion of acids decreased. The epicuticular wax layer also contained alpha-tocopherol described for the first time to be present also in the epicuticular wax. The modifications in the chemical composition of cuticular waxes are discussed in relation to the varying physical characteristics of the cuticle during ontogenesis of apple leaves.     

Nanotubules on plant surfaces: chemical composition of epicuticular wax crystals on needles of Taxus baccata L

Needles of Taxus baccata L. were covered with tubular epicuticular wax crystals varying in diameters (100 and 250 nm) and lengths (300-500 and 500-1000 nm) on the abaxial and adaxial surfaces, respectively. Various sampling protocols were employed to study the chemical composition of the needle waxes on three different levels of spatial resolution. First, a dipping extraction of whole needles yielded the total cuticular wax mixture consisting of very long chain fatty acids (21%), alkanediols (19%), phenyl esters (15%), and secondary alcohols (9%) together with small amounts of aldehydes, primary alcohols, alkanes, alkyl esters, and tocopherols. Second, waxes from both sides of the needle were sampled separately by brushing with CHCl3-soaked fabric glass. Both sides showed very similar qualitative composition, but differed drastically in quantitative aspects, with nonacosan-10-ol (18%) and alkanediols (33%) dominating the abaxial and adaxial waxes, respectively. Third, the epi- and intracuticular wax layers were selectively sampled by a combination of mechanical wax removal and brushing extraction. This provided direct evidence that the tubular wax crystals contained high percentages of nonacosane-4,10-diol and nonacosane-5,10-diol on the abaxial surface, and nonacosan-10-ol on the adaxial surface of the needles. Together with these compounds, relatively large amounts of fatty acids and smaller percentages of aldehydes, primary alcohols, alkyl esters, and alkanes co-crystallized in the epicuticular layer. In comparison, the intracuticular wax consisted of higher portions of cyclic constituents and aliphatics with relatively high polarity. The formation of the tubular crystals is discussed as a spontaneous physico-chemical process, involving the establishment of gradients between the epi- and intracuticular wax layers and local phase separation.     

Cuticular lipids of insects. VI. Cuticular lipids of the grasshoppers Melanoplus sanguinipes and Melanoplus packardii

The cuticular lipids of the grasshoppers Melanoplus sanguinipes and Melanoplus packardii contain 60 and 68% alkanes and 28 and 18% secondary alcohol wax esters, respectively, with lesser amounts of normal and sterol wax esters, triglycerides, alcohols, sterols, and free fatty acids. All the hydrocarbons are saturated, and four types of alkanes are present: n-alkanes, 3-methylalkanes, internally branched monomethylalkanes, and internally branched dimethylalkanes. The principal n-alkanes in both insects are C(29) and C(27), with a range from C(21) to C(33). Trace amounts of 3-methylalkanes of 28, 30, and 32 total carbons are present. The principal internally branched monomethylalkanes are C(32) and C(34), whereas the main dimethylalkane contains 35 carbons. The n-alkanes do not correspond in chain length to the secondary alcohols. The primary alcohols range from C(22) to C(32) in both insects, with C(24) and C(26) predominating. The fatty acids in the triglyceride and free fatty acid fractions range from C(12) to C(24) in M. sanguinipes and from C(12) to C(18) in M. packardii.     

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.     

The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration barrier properties: effects of a deficiency in a beta-ketoacyl-coenzyme A synthase (LeCER6)

Cuticular waxes play a pivotal role in limiting transpirational water loss across the primary plant surface. The astomatous fruits of the tomato (Lycopersicon esculentum) 'MicroTom' and its lecer6 mutant, defective in a beta-ketoacyl-coenzyme A synthase, which is involved in very-long-chain fatty acid elongation, were analyzed with respect to cuticular wax load and composition. The developmental course of fruit ripening was followed. Both the 'MicroTom' wild type and lecer6 mutant showed similar patterns of quantitative wax accumulation, although exhibiting considerably different water permeances. With the exception of immature green fruits, the lecer6 mutant exhibited about 3- to 8-fold increased water loss per unit time and fruit surface area when compared to the wild type. This was not the case with immature green fruits. The differences in final cuticular barrier properties of tomato fruits in both lines were fully developed already in the mature green to early breaker stage of fruit development. When the qualitative chemical composition of fruit cuticular waxes during fruit ripening was investigated, the deficiency in a beta-ketoacyl-coenzyme A synthase in the lecer6 mutant became discernible in the stage of mature green fruits mainly by a distinct decrease in the proportion of n-alkanes of chain lengths > C(28) and a concomitant increase in cyclic triterpenoids. This shift in cuticular wax biosynthesis of the lecer6 mutant appears to be responsible for the simultaneously occurring increase of water permeance. Changes in cutin composition were also investigated as a function of developmental stage. This integrative functional approach demonstrates a direct relationship between cuticular transpiration barrier properties and distinct chemical modifications in cuticular wax composition during the course of tomato fruit development.     

Diversity of cuticular wax among Salix species and Populus species hybrids

The leaf cuticular waxes of three Salix species and two Populus species hybrids, selected for their ability to produce high amounts of biomass, were characterized. Samples were extracted in CH(2)Cl(2) three times over the growing season. Low kV SEM was utilized to observe differences in the ultrastructure of leaf surfaces from each clone. Homologous series of wax components were classified into organic groups, and the variation in wax components due to clone, sample time, and their interaction was identified. All Salix species and Populus species hybrids showed differences in total wax load at each sampling period, whereas the pattern of wax deposition over time differed only between the Salix species. A strong positive relationship was identified between the entire homologous series of alcohols and total wax load in all clones. Similarly strong relationships were observed between fatty acids and total wax load as well as fatty acids and alcohols in two Salix species and one Populus species hybrid. One Salix species, S. dasyclados, also displayed a strong positive relationship between alcohols and alkanes. These data indicate that species grown under the same environmental conditions produce measurably different cuticular waxes and that regulation of wax production appears to be different in each species. The important roles cuticular waxes play in drought tolerance, pest, and pathogen resistance, as well as the ease of wax extraction and analysis, strongly suggest that the characteristics of the cuticular wax may prove to be useful selectable traits in a breeding program.     

Waxes and lipids associated with the external waxy structures of nymphs and pupae of the giant whitefly, Aleurodicus dugesii

The nymphs and pupae of the giant whitefly, Aleurodicus dugesii, produce large quantities of external lipids, both as waxy particles and as waxy filaments. The nymphs and pupae extrude filaments from two dorsal rows of five pores each. Filaments can attain lengths of 5-8 cm. The external lipids of nymphs and pupae consist largely of long-chain aldehydes, alcohols, acetate esters and wax esters. Hydrocarbons are minor components. Soon after hatching, the nymph produced an unidentified waxy fringe extruded laterally from its margin. After molting to the second instar, long, hollow, waxy filaments were produced by the immature stages. The major lipid class associated with the filaments was saturated wax esters (89%), mainly C44, C46 and C60. Associated with formation of the filaments were waxy particles in the shape of curls, which peeled off of the extruding filaments. Similar but more tubular-shaped curls were also produced by numerous lateral pores so that, eventually, the curls completely camouflaged the nymph. The major lipid class of the curls was wax esters (50%), mainly C44 and C46. The cuticular surface lipids of the nymphs were mainly long-chain aldehydes (43%) and wax esters (27%). Unsaturated fatty acid moieties constituted 2 and 19% of the wax esters of curls and nymph cuticular surface lipids, respectively. The major lipid classes of pupae and of their palisade were long-chain aldehydes and alcohols. No unsaturated wax esters were detected in the filaments, but 30% of pupal and 21% of palisade surface wax esters were unsaturated in their fatty acid moieties, 16:1, 18:1 and 20:1.     

Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development

The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 microg leaf(-1) of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.     

The effects of Kloeckera apiculata on the cuticular waxes of navel orange fruit

Kloeckera apiculata 34-9 was selected from the rhizosphere soil for its high efficacy in controlling citrus green and blue mold. In this study, the effect of the antagonistic yeast K. apiculata on citrus cuticular wax was investigated in ripening Newhall navel orange (Citrus sinensis L. Osbeck). Our results show that K. apiculata triggers the production of cuticular waxes and surface wax morphology changes in the fruit surface. 15 K. apiculata-responsive differentially expressed genes (DEGs) were identified for wax metabolism by using the Affymetrix citrus genome GeneChip. Using GC–MS, 46 wax compounds were found in the Newhall fruit surface. On one hand, esters including docosanoic acid, 1,2,3-propanetriyl ester and 9-hexadecenoic acid, 9-octadecenyl ester were up-regulated approximately twofold in the treatment condition compared with the control; and on the other hand, the fatty acids and fatty alcohols decreased by 74.4% and 72.3%, respectively, in the treatment condition. The new wax production and increased hydrophobicity of the Newhall surface resulting from the treatment may influence spore adhesion and germination. Furthermore, the response of the fruit waxes to the K. apiculata stimulus is likely to be regulated by intra-cellular H₂O₂ signaling. This study demonstrated the response fruit waxes to K. apiculata in Newhall navel oranges, thus providing new clues that aid our understanding of the mechanisms of action of antagonistic yeasts in postharvest fruits.     

A comparison of the composition of epicuticular wax from red raspberry (Rubus idaeus L.) and hawthorn (Crataegus monogyna Jacq.) flowers

Epicuticular waxes have been characterised from the flowers of raspberry and hawthorn, on both of which adult raspberry beetles (Byturus tomentosus) can feed. The flower wax from both species had similar alkane profiles and also contained long-chain alcohols, aldehydes and fatty acids. The range of the carbon numbers detected for these classes of compounds was broadly similar in both but the relative amounts of each differed between species. Raspberry flower wax also contained fatty acid methyl esters, a group of compounds that has rarely been detected in plant epicuticular waxes, however, these were not observed in hawthorn flower wax. Long-chain alcohol-fatty acid esters with carbon numbers ranging from C36 to C48 were also detected in both plant species. However, an examination of their constituent acids indicated that in hawthorn the esters based on the C16 fatty acid predominated, whilst in raspberry flower wax, esters based on the C20 fatty acid were most abundant. Both species also contained pentacyclic triterpenoids, which accounted for, on average, over 16 and 48% of the total wax extracted from raspberry and hawthorn flowers respectively. In the former, ursolic and oleanolic acids accounted for over 90% of the pentacyclic triterpenes, whilst hawthorn flower wax, in addition to containing these acids, also contained high relative concentrations of both free and esterified alpha- and beta-amyrins.     

Monoacylglycerols are components of root waxes and can be produced in the aerial cuticle by ectopic expression of a suberin-associated acyltransferase

The interface between plants and the environment is provided for aerial organs by epicuticular waxes that have been extensively studied. By contrast, little is known about the nature, biosynthesis, and role of waxes at the root-rhizosphere interface. Waxes isolated by rapid immersion of Arabidopsis (Arabidopsis thaliana) roots in organic solvents were rich in saturated C18-C22 alkyl esters of p-hydroxycinnamic acids, but also contained significant amounts of both alpha- and beta-isomers of monoacylglycerols with C22 and C24 saturated acyl groups and the corresponding free fatty acids. Production of these compounds in root waxes was positively correlated to the expression of sn-glycerol-3-P acyltransferase5 (GPAT5), a gene encoding an acyltransferase previously shown to be involved in aliphatic suberin synthesis. This suggests a direct metabolic relationship between suberin and some root waxes. Furthermore, when ectopically expressed in Arabidopsis, GPAT5 produced very-long-chain saturated monoacylglycerols and free fatty acids as novel components of cuticular waxes. The crystal morphology of stem waxes was altered and the load of total stem wax compounds was doubled, although the major components typical of the waxes found on wild-type plants decreased. These results strongly suggest that GPAT5 functions in vivo as an acyltransferase to a glycerol-containing acceptor and has access to the same pool of acyl intermediates and/or may be targeted to the same membrane domain as that of wax synthesis in aerial organs.     

Cuticular waxes from ivy leaves (Hedera helix L.): analysis of high-molecular-weight esters

Soluble waxes were extracted from the cuticle of ivy (Hedera helix L.) leaves with dichloromethane in a yield of ca. 13%. The cuticular waxes were directly analysed by GC-MS, high-temperature GC-MS and ESI-MS/MS. The GC-MS analysis showed mostly n-alkanols (45.3%), monoacids (18.8%), triterpenes (9.7%), n-aldehydes (8.7%) and n-alkanes (7.7%). The high-temperature GC-MS and the ESI-MS/MS analyses showed the presence of ester waxes, namely alkyl alkanoates and alkyl coumarates. Alkyl alkanoates comprised esters of the hexadecanoic acid with n-alkanols ranging from C16 to C34. Alkyl coumarates included esters of coumaric acid with n-alkanols ranging from C16 to C32. The cuticular waxes were hydrolysed and the resulting organic and aqueous phases analysed by GC-MS. The hydrolysate showed a major increase in the quantities of n-alkanols, hexadecanoic acid and coumaric acid derived from the alkyl and acyl moieties from the ester waxes. A content of ester waxes of 38% was estimated based on the results from the GC-MS analysis of the non-hydrolysed and hydrolysed cuticular waxes. Alkyl alkanoates were analysed by ESI-MS/MS as [M + Li]+ adduct ions and the alkyl coumarates as [M - H]- deprotonated ions. The ESI-MS/MS analysis allowed the detection of a wider range of ester waxes than high-temperature GC-MS, and was shown to be a useful technique for the qualitative analysis of ester waxes from plant cuticles.     

Composition of sugarcane waxes in rum factory wastes

Wastes produced during fermentation and distillation of crude sugarcane juice in rum factories were evaluated as a new source of waxes. The chemical composition of the crude wax extracted from adsorbat of the wastes on fuller's earth was studied by GC-mass spectrometry. Series of linear alkanes (C19-C33), and wax esters constitute the main components. In addition, phytosterols, triterpene methyl ethers, ethyl and methyl esters of fatty acids, and free fatty acids were found as minor components. Acid (predominance of C16 and C18) and alcohol portions (C26-C32) of the wax esters were analysed after saponification.     

Crystal structures and chemical composition of leaf surface wax depositions on the desert moss Syntrichia caninervis

The structures and composition of cuticular waxes deposited on the leaves of a typical desert moss, Syntrichia caninervis, were investigated. The wax crystals deposited on leaves shifted with leaf aging. The results of chemical analysis showed the main chemical components of the moss wax were fatty acids, alcohols and alkanes. Leaf aging increased the content of cuticular wax and the percentage of very long chain components, from 1150 μg g⁻¹ DW and 13.6% in younger leaves to 2640 μg g⁻¹ DW and 37.2% in aged leaves, respectively. Dehydration/hydration also augmented the wax content by 35.17% in juvenile leaves and by 1900% in lab-cultivated leaves after three-cycle treatments. Synthesis of hexadecanoic acid and tetracosane were predicted to be the first step of wax accumulation. The responses of cuticular waxes in crystal structure and chemical composition were recommended as a biomonitor for assessing the shift of ecological and environmental quality.     

Novel wax esters and hydrocarbons in the cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus

The cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus, were found to contain minor amounts of novel wax esters, in addition to the major components, hydrocarbons. The wax esters ranged in carbon number from C19 to C31 and consisted of esters of both odd- and even-numbered alcohols and acids. Each wax ester with a given carbon number eluted at several different retention times indicating possible methyl branching in either the fatty acid or alcohol moiety, or in both moieties. Each eluting peak of wax esters consisted of a mixture of wax esters of the same carbon number in which the fatty acid moiety ranged from C8 to C18, and the alcohol moiety ranged from C8 to C17. Some wax esters were largely found on the head indicating they may be of a glandular origin. The hydrocarbons consisted of: n-alkanes, C23 to C33; odd-numbered n-alkenes, C27 to C35; and the major components, methyl-branched alkanes, C26 to over C49. Notable components of the methyl-branched alkanes were 2-methyltriacontane, and the novel trimethylalkanes with a single methylene between the first and second branch points, 13,15,19-trimethylhentriacontane and 13,15,21-trimethyltritriacontane.     

Leaf sheath cuticular waxes on bloomless and sparse-bloom mutants of Sorghum bicolor

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.