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.     

Fruit cuticle lipid composition and water loss in a diverse collection of pepper (Capsicum)

Pepper (Capsicum spp.) fruits are covered by a relatively thick coating of cuticle that limits fruit water loss, a trait previously associated with maintenance of postharvest fruit quality during commercial marketing. To shed light on the chemical‐compositional diversity of cuticles in pepper, the fruit cuticles from 50 diverse pepper genotypes from a world collection were screened for both wax and cutin monomer amount and composition. These same genotypes were also screened for fruit water loss rate and this was tested for associations with cuticle composition. Our results revealed an unexpectedly large amount of variation for the fruit cuticle lipids, with a more than 14‐fold range for total wax amounts and a more than 16‐fold range for cutin monomer amounts between the most extreme accessions. Within the major wax constituents fatty acids varied from 1 to 46%, primary alcohols from 2 to 19%, n‐alkanes from 13 to 74% and triterpenoids and sterols from 10 to 77%. Within the cutin monomers, total hexadecanoic acids ranged from 54 to 87%, total octadecanoic acids ranged from 10 to 38% and coumaric acids ranged from 0.2 to 8% of the total. We also observed considerable differences in water loss among the accessions, and unique correlations between water loss and cuticle constituents. The resources described here will be valuable for future studies of the physiological function of fruit cuticle, for the identification of genes and QTLs associated with fruit cuticle synthesis in pepper fruit, and as a starting point for breeding improved fruit quality in pepper.     

Cutins of Malus pumila fruits and leaves

The cutins of fruits and leaves of four apple cultivars have been analysed using TLC, GLC and GC-MS. They are similarly composed of saturated, monounsaturated and diunsaturated fatty, hydroxy-fatty and epoxyhydroxy-fatty acids. The most abundant monomers are 18-hydroxyoctadeca-9,12-dienoic, 10,16-dihydroxyhexadecanoic, 9,10-epoxy-18-hydroxyoctadec-12-enoic, 9,10-epoxy-18-hydroxyoctadecanoic and 9,10,18-trihydroxyoctadecanoic acids. The fruit cutins have high contents of epoxides (35–40%) and unsaturated components ( > 40%) and C₁₈ compounds predominate over C₁₆. The leaf cutins contain smaller amounts of unsaturated components than the fruits and higher proportions of C₁₆ compounds. The adaxial leaf cutin differs in composition from the abaxial. 10,16-Dihydroxyhexadecanoic and 9,10-epoxy-18-hydroxoctadecanoic acids are the major constituents (each ca. 30%) of the adaxial leaf cutin and 10,16-dihydroxyhexadecanoic acid (55–65%) predominates in the abaxial.     

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8 / LACS1 , one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C₂₄) were elevated more than 155%. The C₁₆ cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C₁₈ monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C₂₀–C₃₀, with highest activity for C₃₀ acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C₁₆ but not C₁₈ cutin monomers are reduced in lacs1 , and C₁₆ acids are the next most preferred acid (behind C₃₀) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C₁₆ monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C₁₆) fatty acids for cutin synthesis.     

The constituent cutin acids of cranbury cuticle

Purified cutin from cranberry (Vaccinium macrocarpon, var. Howes) skin was selectively degraded, and the cutin acids, as methyl esters, separated by TLC into seven classes including monobasic acids, dibasic acids, monohydroxy monobasic acids, monohydroxy epoxymonobasic acids, vic-dihydroxy dibasic acids, dihydroxy monobasic acids and trihydroxy monobasic acids. Of the 41 components identified in cranberry cutin by GLC and MS analysis, 18-hydroxyoctadec-cis-9-enoic acid (9·4%), 18-hydroxy-cis-9,10-epoxyoctadecanoic acid (7·5%), 10,16-dihydroxyhexadecanoic acid (16·7%) and threo-9,10,18-trihydroxyoctadecanoic acid (43·7%) were shown to be the major constituents.     

Fruit cuticle lipid composition and fruit post-harvest water loss in an advanced backcross generation of pepper (Capsicum sp.)

To understand the role of fruit cuticle lipid composition in fruit water loss, an advanced backcross population, the BC(2)F(2) , was created between the Capsicum annuum (PI1154) and the Capsicum chinense (USDA162), which have high and low post-harvest water loss rates, respectively. Besides dramatic differences in fruit water loss, preliminary studies also revealed that these parents exhibited significant differences in both the amount and composition of their fruit cuticle. Cuticle analysis of the BC(2)F(2) fruit revealed that although water loss rate was not strongly associated with the total surface wax amount, there were significant correlations between water loss rate and cuticle composition. We found a positive correlation between water loss rate and the amount of total triterpenoid plus sterol compounds, and negative correlations between water loss and the alkane to triterpenoid plus sterol ratio. We also report negative correlations between water loss rate and the proportion of both alkanes and aliphatics to total surface wax amount. For the first time, we report significant correlations between water loss and cutin monomer composition. We found positive associations of water loss rate with the total cutin, total C(16) monomers and 16-dihydroxy hexadecanoic acid. Our results support the hypothesis that simple straight-chain aliphatic cuticle constituents form more impermeable cuticular barriers than more complex isoprenoid-based compounds. These results shed new light on the biochemical basis for cuticle involvement in fruit water loss.     

Aliphatic composition of cutin from inner seed coat of apple

Lithium aluminum deuteride reduction released aliphatic monomers from the inner seed coat fraction but not from the outer seed coat fraction of mature apples. These monomers were identified by GC/MS and the results indicate that the inner coat of apple seed contains a cutin polymer with the major monomer acids being 18-hydroxyoctadec-9-enoic (31%), 9,10-epoxy-18-hydroxyoctadecanoic (28%) and 9,10,18-trihydroxyoctadecanoic (20 %). The monomer composition of this seed coat cuticular polymer was very similar in seeds taken from freshly harvested fruit and in those taken from fruit which had been stored at 4° for 6 months.     

The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds

Mature seeds of Arabidopsis thaliana and Brassica napus contain a complex mixture of aliphatic monomers derived from the non-extractable lipid polyesters deposited by various seed tissues. Methods of polyester depolymerization of solvent-extracted seeds and analysis of aliphatic monomers were compared. Sodium methoxide-catalyzed depolymerization, followed by GC analysis of the acetylated monomers, was developed for routine quantitative analysis suitable for 0.5g seed samples. In Arabidopsis seeds, the major C16 and C18 monomers identified included omega-hydroxy fatty acids and alpha,omega-dicarboxylic acids derived from palmitate, oleate and linoleate, and 9,10,18-trihydroxyoctadecenoic acid. Among monomers which can collectively be considered likely to be derived from suberin, docosan-1-ol, docosane-1,22-diol, 22-hydroxydocosanoic acid, 24-hydroxytetracosanoic acid, tetracosane-1,24-dioic acid and ferulic acid were the major species. Compared to Arabidopsis, Brassica seeds showed a roughly similar proportion of monomer classes, with the exception that alkan-1ols were 3-fold higher. Also, there were much less C24 aliphatic species and significant amounts of C14-C16 alkan-1ols, including iso- and anteiso-methyl branched compounds. Dissection and analysis of mature Brassica seeds showed that the trihydroxy C18:1 fatty acid was found mainly in the embryo, while ferulate, fatty alcohols and C22 and C24 species were specific to the seed coat plus endosperm.     

Studies on water transport through the sweet cherry fruit surface: II. Conductance of the cuticle in relation to fruit development

Water conductance of the cuticular membrane (CM) of sweet cherry (Prunus avium L. cv. Sam) fruit during stages II and III (31-78 days after full bloom, DAFB) was investigated by gravimetrically monitoring water loss through segments of the exocarp. Segments were mounted in stainless-steel diffusion cells, filled with 0.5 ml of deionized water and incubated for 8 h at 25 +/- 2 degrees C over dry silica. Conductance was calculated by dividing the amount of water transpired per unit surface area and time by the difference in water vapor concentration across the segment (23.07 g m(-3) at 25 degrees C). Fruit mass and fruit surface area increased 4.9- and 2.8-fold between 31 and 78 DAFB, respectively. However, CM mass per unit area decreased from 3.9 to 1.5 g m(-2) and percentage of total wax content remained constant at about 31%. Stomatal density decreased from 0.8 to 0.2 mm(-2) (31-78 DAFB). Total conductance of the CM on the fruit cheek (gtot.) remained constant during stage II of development (approx. 1.38 x 10(-4) m s(-1) from 31 to 37 DAFB), increased to 1.73 x 10(-4) m s(-1) during early stage III of fruit growth (43-64 DAFB) then decreased to 0.95 x 10(-4) m s(-1) at maturity (78 DAFB). Partitioning gtot. into cuticular (gcut.) and stomatal conductance (gsto.) revealed that the relative contribution of gcut. to gtot. increased linearly from 30% to 87% of gtot. between 31 and 78 DAFB. respectively. On a whole-fruit basis, g,tot. and gcut. consistently increased up to 64 DAFB, and decreased thereafter. A significant negative linear relationship was obtained between gcut. and CM thickness, but not between the permeability coefficient (p) and CM thickness. Further, p was positively related to strain rate, suggesting that strain associated with expansion of the fruit surface increased p.     

Changes in strain and deposition of cuticle in developing sweet cherry fruit

Changes in surface area, deposition and elastic strain of the cuticular membrane (CM) were monitored during development of sweet cherry (Prunus avium L.) fruit. Fruit mass and surface area ('Sam') increased in a sigmoidal pattern between 16 and 85 days after full bloom (DAFB) with maximum rates of 0.35 g day(-1) and 0.62 cm(2) day(-1), respectively. Rates of total area strain, namely the sum of elastic plus plastic strain, were highest in cheek and stem cavity regions followed by stylar and suture regions. Rates of total uniaxial strain were higher in transverse, namely perpendicular to the stem/stylar axis, than in longitudinal direction, namely parallel to the stem/stylar axis. On a whole fruit basis CM mass remained essentially constant during fruit development. Mass of CM, dewaxed CM and wax per unit surface area decreased during development, particularly between 43 and 71 DAFB. There was no change in wax content of isolated CM. Up to 43 DAFB the surface area of isolated CM was similar to the area prior to excision indicating little elastic strain, but markedly decreased thereafter. Calculating elastic and plastic components of total strain of the CM revealed, that initial deformation up to 22 to 43 DAFB was mostly plastic. Thereafter, elastic strain was evident and both, elastic and plastic deformation, increased linearly with an increase in total strain. There was no consistent difference in the relative contribution of elastic strain to total strain between transverse and longitudinal directions, but both total and elastic strain were larger in the transverse direction. Abrading the CM had only little effect on fruit turgor. However, turgor decreased when the exocarp was cut indicating that the exocarp provided a significant structural shell of a mature sweet cherry fruit ('Regina'). Our data demonstrate, that (1) surface area expansion in sweet cherry fruit causes elastic and plastic strain of the CM, and (2) the onset of elastic strain coincided with the cessation of CM formation.     

Constituent acids of Pinus radiata stem cutin

Stem cutin from P. radiata seedlings grown under winter and summer environmental conditions comprised n-alkanoic, (C₁₀–C₂₆), α, ω-alkanedioic (C₁₄–C₂₂), ω-hydroxyalkanoic (C₁₂–C₂₄), hydroxy-α, ω-alkanedioic and polyhydroxyalkanoic acids. 9-Hydroxyheptadecane-1, 17-dioic, 9-hydroxyoctadecene-1, 18-dioic, 9-hydroxynonadecane-1, 19-dioic, and 10, 17-dihydroxyheptadecanoic acids are newly-identified constituents of gymnosperm cutin. Cutin grown under winter temperatures and photoperiod contained twice the amount of 9, 16-dihydroxyhexadecanoic acid than that in summer-grown cutin, suggesting that the winter-grown cutin was formed from a highly cross-linked polymer, and that summer-grown cutin contained more linear polyester portions in the polymer.     

The composition of suberin from the corks of Quercus suber L. and Betula pendula roth

The suberin constituents of Quercus suber and Betula pendula have been isolated after alkaline hydrolysis of the corks and over 80% by weight identified using thin-layer chromatography, preparative thin-layer chromatography, gas-liquid chromatography and combined gas chromatography — mass spectrometry. Long-chain aliphatic acids ranging from C₁₆–C₂₆ comprise about 90% of both suberin fractions; monobasic, α,ω-dibasic, ω-hydroxymonobasic, dihydroxymonobasic, dihydroxydibasic and trihydroxymonobasic acid classes are present. The principal suberin acids of Q. suber are 18-hydroxyoctadecenoic (12%), 22-hydroxydocosanoic (25%), 9,10-dihydroxyoctadecane-1,18-dioic (15%) and 9,10,18-trihydroxyoctadecanoic (8%), and those of B. pendula 9,10,18-trihydroxyoctadecanoic (43%) and 22-hydroxydocosanoic (16%).     

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.     

The cuticles of some Angiosperm leaves and fruits

The cuticles of twenty-four species from a wide range of mono- and di-cotyledonous plants were examined by chemical methods. The cuticles differ markedly in the amount and composition of the surface wax, in the thickness of the cuticular membrane, and in the content and composition of the cutin of the membrane. Fruits usually have heavier wax deposits and much thicker membranes than leaves. No direct relationship exists between surface waxiness and thickness of the membrane. Alkanes and primary alcohols are prominent in many of the surface waxes; triterpenoids occur less frequently. The cutin content of the membrane varies considerably; a delicate membrane tends to have a low content of cutin in which fatty acids are prevalent, and a well-developed membrane a higher content of cutin more rich in hydroxy-fatty acids. 10,16-Dihydroxyhexadecanoic acid is often an important constituent of cutin; 9,10,18-trihydroxyoctadecanoic acid is most prominent in the cutin of thicker membranes. The possible influence of the variations in cutin acids upon the structure of cutin and the taxonomic implications of wax and cutin composition are discussed.     

Suberins of Malus pumila stem and root corks

The suberin contents of the isolated superficial cork layers of Malus pumila stems and root ranged from 15 to 35% of the dry weight. The qualitative composition of the aliphatic monomers obtained after alkaline depolymerization of the extractive-free corks was similar but some quantitative differences were found according to cultivar and age of the cork layer. 1-Alkanols (mainly 22:0, 24:0 and 26:0), alkanoic acids (mainly 22:0 and 24:0), α, ω-alkanedioic acids (mainly 16:0, 18:1 (9) and 18:0) and ω-hydroxyalkanoic acids (mainly 18:1 (9) and 22:0) were major constituents of all the samples examined and together they comprised 40–50% of the total monomeric mixture. The remainder was composed mainly of 9,10-epoxy-18-hydroxy-and 9,10,18-trihydroxyoctadecanoic acids. The corresponding dibasic acids, 9,10-epoxy- and 9,10-dihydroxyoctadecane-1,18-dioic, were minor components as were C₁₆ and C₁₈ dihydroxyalkanoic acids (mainly 10,16-dihydroxyhexadecanoic and 10,18-dihydroxyoctadecanoic acids, respectively). The root suberin dittered from that of the stem in containing larger amounts of 9,10-epoxy- 18-hydroxyoctadecanoic and 18-hydroxyoctadec-9-enoic acids.     

Composition of epicuticular wax layer of two species of Mandevilla (Apocynoideae,Apocynaceae) from Rio de Janeiro,Brazil

The chemical composition of epicuticular waxes of Mandevilla guanabarica and Mandevilla moricandiana was comparatively analyzed by extraction in n-hexane and chloroform. The mean wax content per unit of leaf area in the n-hexane extract was about 13–30 μg cm⁻² for M. guanabarica, containing 20–28% n-alkanes and 55–63% triterpenes; for M. mori-candiana, the mean content was 19 μg cm⁻², containing 73% n-alkanes and 14% triterpenes. In the chloroform extract, the wax yield was 40–80 μg cm⁻² for M. guanabarica, with about 9–11% n-alkanes and 75–82% triterpenes; while for M. moricandiana, the wax yield was 110 μg cm⁻², with 52% n-alkanes and 14% triterpenes. The major compounds identified were lupeol, pentacyclic triterpenes of the α- and β-amyrin class, and n-alkanes such as nonacosane, hentriacontane and tritriacontane. These results indicate that the quantitative chemical profiles of epicuticular waxes of M. guanabarica and M. moricandiana are distinct and could be used as an additional feature in taxonomic identification.     

Biosynthesis of cutin monomers: involvement of a lipoxygenase/peroxygenase pathway

Cutin is synthesized from oxygenated fatty acids derived preponderantly from oleic acid. The enzymatic pathways involved in the biosynthesis of such cutin monomers have been studied, i.e. 18-hydroxyoleic acid, 9,10-epoxy-18-hydroxystearic acid (the major constituent) and 9,10,18-trihydroxystearic acid. This was approached by studying (i) the substrate specificity and stereoselectivity of purified peroxygenase, which epoxidizes unsaturated fatty acids, and fatty acid epoxide hydrolase, i.e. two enzyme activities that have been found recently in higher plants, and (ii) the transformation of oleic acid into cutin monomers by a cell free system, i.e. soybean microsomes. These two enzymes, along with a ω-hydroxylating activity, can account for the biosynthesis of the oleic acid-derived cutin monomers and their precursors. A new biosynthetic scheme is proposed, whose pathways take into account the dynamic aspects of the expression of the different enzyme activities involved. Importantly, since peroxygenase, for its activity, is strictly dependent on fatty acid hydroperoxides, which act as co-substrates, the biosynthesis of cutin monomers is also dependent on the activity of lipoxygenases.     

Composition,ultrastructure and function of the cutin- and suberin-containing layers in the leaf,fruit peel,juice-sac and inner seed coat of grapefruit (Citrus paradisi Macfed.)

Cutin and suberin polymers from various anatomical regions of grapefruit were analyzed chemically and ultrastructurally. The leaf, fruit peel and juice-sac showed an amorphous cuticular layer. The cutin in the leaf was composed of 10,16-dihydroxy C₁₆ acid and its positional isomers as the major monomers whereas 16-hydroxy-10-oxo C₁₆ acid was a major component in the fruit peel. Juice-sac cutin, on the other hand, contained the dihydroxy C₁₆ acids, hydroxyoxo C₁₆ acids, hydroxyepoxy C₁₈ acids and trihydroxy C₁₈ acids. Ultrastructural examination of the inner seed coat showed that an amorphous cuticular layer encircled the entire seed except in the chalazal region which showed several layers of cells with lamellar suberin structure throughout the cell walls. Consistent with the ultrastructural assignment, the compositions of the aliphatic components of the polymers from the chalazal region and the non-chalazal region indicated the presence of suberin and cutin, respectively. The aliphatic portion of the polymer from the chalazal region of the inner seed coat contained C₁₆, C_18:1, C₂₂ and C₂₄ -hydroxy acids (46% combined total) and the corresponding dicarboxylic acids (43%) as the major components. -Hydroxy-9,10-epoxy C₁₈ acids and 9,10,18-trihydroxy C₁₈ acids were the major components (77%) of the polymer from the non-chalazal portion of the inner seed coat. The main portion and the chalazal region of the inner seed coat yielded 17 and 342 g/cm² of aliphatic monomers, respectively, and the diffusion resistance of these two portions of the inner seed coat were 62 and 192 sec/cm, respectively. The inner seed coat was shown to be the major moisture diffusion barrier influencing imbibition and germination.Scientific Paper No. 5649, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164