Cutin Components Research Articles

Microbial degradation of poly(ε-caprolactone) in a coastal environment

Biodegradable plastics may be a solution for eliminating large marine plastic debris, such as fishing gear, which causes environmental damage. The biodegradable polyester poly(ε-caprolactone) (PCL) is degraded in various environments; however, no reports have examined PCL-degrading microbes inhabiting coastal environments, despite the fact that marine plastic debris accumulates in this environment. Therefore, in this study, we isolated a PCL-degrading bacterium (strain TKCM 64) from marine plastic debris in coastal seawater (Okinoshima Park, Chiba, Japan) for the first time. Genetic and biochemical analyses showed that strain TKCM 64 was closely related to Pseudomonas pachastrellae JCM12285T. The maximum specific growth rate (0.4 h−1) of the isolate was observed in the absence of NaCl at 30 °C, and the strain was able to grow, even at 1.20 M NaCl, suggesting that the strain could belong to a marine species showing halotolerance. Strain TKCM 64 degraded PCL film at a rate of 1.39 ± 0.09 mg cm−2·day−1. The strain showed PCL hydrolytic activity, which was induced in the presence of PCL and its hydrolysate: 6-hydroxyhexanoic acid (6HH), in addition to a major component of cutin, 16-hydroxyhexadecanoic acid. Thus, we concluded that the PCL hydrolysate 6HH, as an analog of cutin hydrolysate, functioned as an inducer of a PCL hydrolase that is a kind of cutinase in strain TKCM 64. In addition, Pseudomonas pachastrellae was found to be a key bacterial species involved in PCL degradation in coastal environments.

Quantitative analysis of glycerol in dicarboxylic acid-rich cutins provides insights into Arabidopsis cutin structure

Cutin is an extracellular lipid polymer that contributes to protective cuticle barrier functions against biotic and abiotic stresses in land plants. Glycerol has been reported as a component of cutin, contributing up to 14% by weight of total released monomers. Previous studies using partial hydrolysis of cuticle-enriched preparations established the presence of oligomers with glycerol-aliphatic ester links. Furthermore, glycerol-3-phosphate 2-O-acyltransferases (sn-2-GPATs) are essential for cutin biosynthesis. However, precise roles of glycerol in cutin assembly and structure remain uncertain. Here, a stable isotope-dilution assay was developed for the quantitative analysis of glycerol by GC/MS of triacetin with simultaneous determination of aliphatic monomers. To provide clues about the role of glycerol in dicarboxylic acid (DCA)-rich cutins, this methodology was applied to compare wild-type (WT) Arabidopsis cutin with a series of mutants that are defective in cutin synthesis. The molar ratio of glycerol to total DCAs in WT cutins was 2:1. Even when allowing for a small additional contribution from hydroxy fatty acids, this is a substantially higher glycerol to aliphatic monomer ratio than previously reported for any cutin. Glycerol content was strongly reduced in both stem and leaf cutin from all Arabidopsis mutants analyzed (gpat4/gpat8, att1-2 and lacs2-3). In addition, the molar reduction of glycerol was proportional to the molar reduction of total DCAs. These results suggest “glycerol-DCA-glycerol” may be the dominant motif in DCA-rich cutins. The ramifications and caveats for this hypothesis are presented.

ABCG15 Encodes an ABC Transporter Protein, and is Essential for Post-Meiotic Anther and Pollen Exine Development in Rice

In flowering plants, anther and pollen development is critical for male reproductive success. The anther cuticle and pollen exine play an essential role, and in many cereals, such as rice, orbicules/ubisch bodies are also thought to be important for pollen development. The formation of the anther cuticle, exine and orbicules is associated with the biosynthesis and transport of wax, cutin and sporopollenin components. Recently, progress has been made in understanding the biosynthesis of sporopollenin and cutin components in Arabidopsis and rice, but less is known about the mechanisms by which they are transported to the sites of deposition. Here, we report that the rice ATP-binding cassette (ABC) transporter, ABCG15, is essential for post-meiotic anther and pollen development, and is proposed to play a role in the transport of rice anther cuticle and sporopollenin precursors. ABCG15 is highly expressed in the tapetum at the young microspore stage, and the abcg15 mutant exhibits small, white anthers lacking mature pollen, lipidic cuticle, orbicules and pollen exine. Gas chromatography-mass spectrometry (GC-MS) analysis of the abcg15 anther cuticle revealed significant reductions in a number of wax components and aliphatic cutin monomers. The expression level of genes involved in lipid metabolism in the abcg15 mutant was significantly different from their levels in the wild type, possibly due to perturbations in the homeostasis of anther lipid metabolism. Our study provides new insights for understanding the molecular mechanism of the formation of the anther cuticle, orbicules and pollen wall, as well as the machinery for lipid metabolism in rice anthers.

Single-solute and bi-solute sorption of phenanthrene and pyrene onto pine needle cuticular fractions

To better understand interaction mechanisms of pine needles with persistent organic pollutants, single-solute and bi-solute sorption of phenanthrene and pyrene onto isolated cuticular fractions of pine needle were investigated. The structures of cuticular fractions were characterized by elemental analysis, Fourier transform infrared spectroscopy and solid-state 13C NMR. Polymeric lipids (cutin and cutan) exhibited notably higher sorption capabilities than the soluble lipids (waxes), while cellulose showed little affinity with sorbates. With the coexistence of the amorphous cellulose, the sorption of cutan (aromatic core) was completely inhibited, so the cutin components (nonpolar aliphatic moieties) dominated the sorption of bulk needle cuticle. By the consumption of the amorphous cellulose under acid hydrolysis, sorption capacities of the de-sugared fractions were dramatically enhanced, which controlled by the exposed aromatic cores and the aliphatic moieties. Furthermore, the de-sugared fractions demonstrated nonlinear and competitive sorption due to the specific interaction between aromatic cores and polycyclic aromatic hydrocarbon.

Expression of glycine-rich protein genes, AtGRP5 and AtGRP23, induced by the cutin monomer 16-hydroxypalmitic acid in Arabidopsis thaliana

Glycine-rich proteins (GRPs) belong to a large family of heterogenous proteins that are enriched in glycine residues. The expression of two GRP genes of Arabidopsis thaliana, AtGRP5 and AtGRP23, was induced by 16-hydroxypalmitic acid (HPA), a major component of cutin. The expression of AtGRP3, which encodes a GRP protein that is structurally different from AtGRP5 and AtGRP23, was not responsive to HPA application. Treatment with HPA also induced expression of the pathogen-related PR-1 and PR-4 genes. Abscisic acid and salicylic acid treatments enhanced the transcript levels of AtGRP5 and AtGRP23 as well as those of AtGRP3. It was also demonstrated that HPA effectively elicited the accumulation of H2O2 in rosette leaves of Arabidopsis. Results suggest the possible role of some species of GRPs, such as AtGRP5 and AtGRP23, in response to the pathogenic invasion mediated by cutin monomers in plants.

Cutin monomer induces expression of the rice OsLTP5 lipid transfer protein gene

Treatment with the cutin monomer 16-hydroxypalmitic acid (HPA), a major component of cutin, elicited the synthesis of hydrogen peroxide (H2O2) in rice leaves and induced the expression of the lipid transfer protein gene OsLTP5. Treatment with HPA also induced expression of OsLTP1, OsLTP2, and the pathogen-related PR-10 genes to a lesser extent. The OsLTP5 transcript was expressed prominently in stems and flowers, but was barely detectable in leaves. Expression of OsLTP5 was induced in shoots in response to ABA and salicylic acid. It is proposed that HPA is perceived by rice as a signal, inducing defense reactions.

Analysis of the aliphatic monomer composition of polyesters associated with Arabidopsis epidermis: occurrence of octadeca‐cis‐6, cis‐9‐diene‐1,18‐dioate as the major component

Although the surface waxes from Arabidopsis thaliana leaves and stems have been thoroughly characterized, the monomer composition of the polyesters of the cuticular membrane has not been analyzed. Delipidated Arabidopsis leaves or stems, when depolymerized under conditions to cleave polyesters, produced typical omega-hydroxy fatty acid cutin monomers such as 16-hydroxy-palmitate, 10,16-dihydroxy-palmitate and 18-hydroxy-9,10-epoxy-stearate. However, the major monomer was octadeca-cis-6, cis-9-diene-1,18-dioate, with lesser amounts of octadec-cis-9-ene-1,18-dioate and hexadeca-1,16-dioate. These dicarboxylates were found predominantly in epidermal peels from Arabidopsis stems and are therefore likely to be associated with the cuticular membrane. They were also found in analyses of canola leaves but were absent in tomato and apple fruit cutins. In the fad2-1 mutant line of Arabidopsis, which has reduced levels of linoleate and linolenate and elevated oleate in cytosolic phospholipids, the amount of octadeca-cis-6, cis-9-diene-1,18-dioate was 50% reduced, with a concomitant increase in octadec-cis-9-ene-1,18-dioate. In a fatb-ko line of Arabidopsis, where the availability of cytosolic palmitate is impaired, there was an 80% loss of C16 monomers and a compensating increase in C18 monomers. The presence of substantial amounts of dicarboxylates in cuticular membranes is unexpected. High amounts of aliphatic dicarboxylates are usually considered as an indicator of suberin, and are reported only as very minor components of cutin. The high level of polyunsaturation is also unusual in cuticles; saturated fatty acid monomers usually predominate, with lesser amounts of monounsaturates. These novel findings for Arabidopsis demonstrate that a broad range of monomer compositions are possible for polyesters of the epidermis.

Liming effects on the chemical composition of the organic surface layer of a mature Norway spruce stand ( Picea abies [L.] Karst.)

The application of lime in a mature Norway spruce ( Picea abies [L.] Karst.) forest in southern Germany induced major changes in the activity of soil organisms and root growth. Since this may influence the chemical compostion of the soil organic matter (SOM) of the organic surface layer, its composition and changes due to the treatment were examined in this study. Fine roots of Norway spruce have a relatively low content of extractable lipids, a low alkyl C content ( 13C CPMAS NMR) and a high ratio of non-cellulosic to cellulosic carbohydrates (NC/CC, carbohydrate determination by MBTH and gas chromatography analyses) as compared to needles. Furthermore, they show high ratios of suberin/cutin compounds (thermally assisted hydrolysis and methylation, (THM)) and high ratios of eicosanic acid/phytadiene I in their lipid extracts (pyrolysis-GC/MS). Liming (4 t ha −1 dolomite) of a Norway spruce organic surface layer decreased the proportion of alkyl C, the alkyl C/ O-alkyl C ratio, and the content of extractable lipids. The NC/CC ratio and the abundance of suberin relative to cutin components increased. The contribution of the chlorophyll component phytadiene I decreased in relation to eicosanic acid. These changes are attributed to increased fine root formation in the organic layer after liming. Furthermore, the presence of less degraded lignin (THM, peak ratio of 3,4-dimethoxybenzoic acid, methyl ester/3,4-dimethoxy-benzaldehyde) on the limed plot is explained by the increased input of relatively fresh fine root material. On the other hand, the decrease in the carbon-to-nitrogen ratio may be attributed to the higher microbial activity after liming.

Characterization and comparison of hydrophobic neutral and hydrophobic acid dissolved organic carbon isolated from three municipal landfill leachates

The acid-precipitated (AP) and acid-soluble (AS) fractions of the combined hydrophobic neutral and hydrophobic acid dissolved organic carbon (DOC) were isolated from leachate collected from three municipal landfills of different age and redox conditions. The AP and the AS combined hydrophobic neutral and hydrophobic acid DOC comprised 6–15% and 51–66%, respectively, of the leachate nonpurgable organic carbon. Elemental analysis, infra-red spectroscopy, 13C CP-MAS nuclear magnetic resonance spectroscopy and dipolar dephasing experiments, and thermochemolysis gas chromatography/mass spectrometry results showed that the AP and AS fractions of hydrophobic neutral and hydrophobic acid DOC are highly aliphatic, with linear and branching moieties, and less oxidized than most terrestrial and aquatic humic substances. Very little, if any, polysaccharide or cellulose, lignin, or cutin components comprise these fractions. It is hypothesized that a majority of the organic carbon in these fractions originates highly branched, cyclic aliphatic organic compounds.

Biochemical analysis of elastic and rigid cuticles of Cirsium horridulum.

The cuticle is a complex structure of soluble lipids, lipid polymers and polysaccharides. In addition to its functions to reduce water loss and provide a protective barrier, its mechanical properties may be significant to plant growth and development. We investigated the cuticle of Cirsium horridulum Michx. because of its involvement in the thigmonastic contraction of staminal filaments. The staminal filaments and portions of the style are surrounded by a highly elastic cuticle in contrast to the rigid cuticle of the corolla and leaves. Our aim was to determine if the biochemical composition affected the elasticity of the cuticle. We discovered that the ratio of carbohydrates to lipids is 1:7 in floral parts but 2:1 in leaf cuticle. Esterified cutin components represented about 80% of the cuticle and di-hydroxyhexadecanoic acids were the major monomers of cutin, regardless of origin. The cutin of elastic tissues is characterized by a higher content of tri-hydroxy monomers than the cutin of rigid tissues. The data suggest that hydroxyl groups enhance the hydrophilic character of the cuticle and contribute to cuticular elasticity.

A comparison of lipid components of the fresh and dead leaves and pneumatophores of the mangrove Avicennia marina

The component hydrocarbons, sterols, alcohols, monobasic, α,ω-dibasic and ω-hydroxy acids of the fresh hand decayed leaves and the pneumatophores of the mangrove Avicennia marina are reported in detail. From the quantitative comparisons which can be drawn, relative changes in the lipid classes occurring during leaf decay can be highlighted. These base-line data are important to our understanding of inputs to marine intertidal sediments. During leaf decay the only significant changes were a reduction in the total absolute concentrations of monobasic acids due largely to a decrease in concentration of the C 18 polyunsaturated fatty acids, and an enhancement of the concentrations of the long-chain monobasic acids, ω-hydroxy acids and α,ω-dibasic acids. This resistance to degradation shown by the cutin derived acids (α,ω-dibasic, ω-hydroxy and long-chain monobasic acids) relative to the cellular and wax derived lipids may allow these cutin components to be used as quantitative markers of A. marina in mangrove associated sediments.

Composition of Lipid-derived Polymers from Different Anatomical Regions of Several Plant Species

The composition of the aliphatics of the protective cuticular polymers from different anatomical regions from several plant species was determined by combined gas-liquid chromatography and mass spectrometry of the depolymerization products derived from the polymers. The polymer from the aerial parts of Vicia faba showed similar composition; dihydroxypalmitic acid was the major (>85%) component of the cutin covering leaves, petioles, flower petals and stem with smaller amounts of palmitic acid and omega-hydroxy palmitic acid. On the other hand, the chief components of the polymer from the tap root were omega-hydroxy C(16:0) and C(18:1) acids and/or the corresponding dicarboxylic acids. The positional isomer composition of the dihydroxy C(16) acids was shown to be dependent upon anatomical location, developmental stage, and light. Apple cutin from rapidly expanding organs (flower petal and stigma) was shown to contain predominately C(16) family acids whereas the C(18) family dominated in cutin of slower growing organs (leaf and fruit). The composition of the aliphatic components of cutin found in the seed coats of pea, corn, barley, and lettuce was found to be similar to that of the cuticular polymer of the leaves in each species.

The optical rotation of a major component of plant cutin.

The major component of cutin from the fruit of both tomato and papaya, dihydroxypalmitate, is shown to have plain positive rotation and is, therefore, assigned L configuration in analogy to other known hydroxy fatty acids.

Midchain hydroxylation of 16-hydroxypalmitic acid by the endoplasmic reticulum fraction from germinating vicia faba

The microsomal fraction from embryonic shoots of germinating Vicia faba catalyzes midchain hydroxylation (at C-9 or C-10) of 16-hydroxypalmitic acid, generating dihydroxypalmitic acid, the major component of cutin, which is the cuticular polymer of higher plants. The hydroxylation product was identified by gas chromatography and reversedphase high-performance liquid chromatography, as well as by gas chromatographic identification of the CrO 3 oxidation products of the hydroxylation product. The midchain hydroxylation required O 2 and NADPH and showed an optimal pH of 7.5. A typical Michaelis-Menton-type substrate saturation pattern was observed for 16-hydroxypalmitic acid, and an apparent K m of 50 μ m was calculated. This hydroxylation was inhibited by NaN 3 , metal ion chelators such as O -phenanthroline and 8-hydroxyquinoline, and thiol-directed reagents such as N -ethylmaleimide and p -chloromercuribenzoate. Photoreversible inhibition of the midchain hydroxylation by CO suggested the involvement of a cytochrome P-450-type protein. Subcellular fractionation of crude extracts using sucrose density gradient centrifugation showed that endoplasmic reticulum is the site of midchain hydroxylation of 16-hydroxypalmitic acid as well as ω-hydroxylation of palmitic acid. Drastic differences in the sensitivity to CO and the photoreversibility of this inhibition suggest that ω-hydroxylation and midchain hydroxylation are catalyzed by two different enzymes.

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/cm 2 of cuticular wax which comprises monobasic acids (C 16–C 38) with hexadecanoic as the major component. The amounts of cutin are comparable with those of cuticular wax and the monomeric constituents are predominantly C 18 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.

Identification of 16-oxo-9-hydroxy hexadecanoic acid a novel monomer, as a major component of the biopolymer cutin in embryonic [formula omitted

16-oxo-9-hydroxy hexadecanoic acid and 16-oxo-10-hydroxy hexadecanoic acid were identified as major components of the cutin of the embryonic shoot of germinating Vicia faba by hydrogenolysis with LiAlH 4, deuterolysis with LiAlD 4 and treatment with NaBD 4 followed by hydrogenolysis and analysis of the products by a combination of gasliquid chromatography and mass spectrometry.

STUDIES ON PLANT CUTICLE: THE COMPOSITION OF THE CUTICLE OF APPLE LEAVES AND FRUITS

The levels of deposition of the waxy and cutin components of the cuticles of the mature leaves and developing fruits of the Merton Worcester and Cox's Orange Pippin varieties of apple over a period of 4 months are reported. The waxy materials on the leaves remain approximately constant at 20 μg./cm.2, and the cutin at about 100 μg./cm.2 of surface. The waxy and cutin deposits on the fruits increase markedly during the period of development, the wax and oil present on the surface and embedded within the cutin rising to 500 μg./cm.2 and the cutin to a little under 1000 μg./cm.2 of surface. Phenolic and acidic substances in the cuticles of the fruits are determined, and the possibility of their occurrence in surface deposits on the leaves is discussed.An examination is made of the effect of nutrient deficiency upon the formation of waxy and phenolic substances in the leaves. Nitrogen deficiency in Worcester Pearmain leaves leads to a reduced deposition of waxy materials, and nitrogen, phosphorus and potassium deficiency results in higher contents of phenolic compounds. The separation and nature of the waxy covering on leaves, the effect of environmental conditions on its formation, and its importance in relation to the application of crop protection chemicals are discussed.