Composition of the cuticle of developing sweet cherry fruit

The composition of wax and cutin from developing sweet cherry (Prunus avium) fruit was studied by GC-MS between 22 and 85 days after full bloom (DAFB). In this and our previous study, fruit mass and surface area increased in a sigmoidal pattern with time, but mass of the cuticular membrane (CM) per unit fruit surface area decreased. On a whole fruit basis, mass of CM increased up to 36 DAFB and remained constant thereafter. At maturity, triterpenes, alkanes and alcohols accounted for 75.6%, 19.1% and 1.2% of total wax, respectively. The most abundant constituents were the triterpenes ursolic (60.0%) and oleanolic acid (7.5%), the alkanes nonacosane (13.0%) and heptacosane (3.0%), and the secondary alcohol nonacosan-10-ol (1.1%). In developing fruit triterpenes per unit area decreased, but alkanes and alcohols remained essentially constant. The cutin fraction of mature fruit consisted of mostly C16 (69.5%) and, to a lower extent, C18 monomers (19.4%) comprising alkanoic, omega-hydroxyacids, alpha,omega-dicarboxylic and midchain hydroxylated acids. The most abundant constituents were 9(10),16-dihydroxy-hexadecanoic acid (53.6%) and 9,10,18-trihydroxy-octadecanoic acid (7.8%). Amounts of C16 and C18 monomers per unit area decreased in developing fruit, but remained approximately constant on a whole fruit basis. Within both classes of monomers, opposing changes occurred. Amounts of hexadecandioic, 16-hydroxy-hexadecanoic, 9(10)-hydroxy-hexadecane-1,16-dioic and 9,10-epoxy-octadecane-1,18-dioic acids increased, but 9,10,18-trihydroxy-octadecanoic and 9,10,18-trihydroxy-octadecenoic acids decreased. There were no qualitative and minor quantitative differences in wax and cutin composition between cultivars at maturity. Our data indicate that deposition of some constituents of wax and cutin ceased during early fruit development.     

Linear aliphatic dimeric esters from cork suberin

Cork suberin was partially depolymerized by methanolysis catalyzed by calcium hydroxide. Analysis by GC-MS of the methanolysate showed suberin monomers, including glycerol and long-chain alpha,omega-diacids and omega-hydroxyacids. ESI-MS analysis of the methanolysate showed, besides the aliphatic monomers, suberin oligomers, including linear dimeric esters of alpha,omega-diacids and omega-hydroxyacids. Two types of dimeric esters were identified: a alpha,omega-diacid linked to a omega-hydroxyacid and two inter-linked omega-hydroxyacids. The alpha,omega-diacids and omega-hydroxyacids found as monomer residues in the dimeric esters were mainly the C18 monomers with midchain substituents. The identification of these dimeric esters was based in their CID-MS/MS spectra and confirmed after synthesis of model compounds. The occurrence of inter-esterified long-chain monomers in suberin brings a new insight in the understanding of the polyester structure of this biopolymer.     

Glycerol derivatives of cutin and suberin monomers: synthesis and self-assembly

Glycerol derivatives of cutin and suberin monomers were synthesized by acid catalysis. Their dispersion in an aqueous solution was examined by phase contrast microscopy, neutron scattering, and solid state NMR. It is shown that the phase behavior strongly depends on the nature of the derivatives forming either lumps of aggregated membranes or well dispersed membranes.     

Evidence for Covalently Attached p-Coumaric Acid and Ferulic Acid in Cutins and Suberins

p-Coumaric acid (4-hydroxycinnamic acid) and ferulic acid (4-hydroxy-3-methoxycinnamic acid) have been identified as constituents of cutin. Their reduction products were isolated from a phenolic fraction released from the cutin of the fruits of apple, peach, pear, and two varieties of tomato and apple leaf by treatment with LiAlH(4) or LiAlD(4). They were identified by combined gas chromatography and mass spectrometry. p-Coumaric acid was present in all samples of cutin (0.07-0.53% by weight), whereas only peach and pear cutin contained measurable amounts of ferulic acid (0.007% and 0.035%, respectively). Both p-coumaric acid and ferulic acid were identified to be constituents of the insoluble material recovered after partial hydrolysis (12-42% loss) of cutin in 1 m NaOH at 80 C. A significant part (48%) of the p-coumaric acid contained in tomato cutin was contained in the insoluble material recovered after partial degradation (7.4%) of this cutin with 0.01 m NaOH. These data indicate that these phenolic components are tightly (possibly covalently) bound to cutin. Similar analysis of the phenolic fractions from the suberins of potato, sweet potato, turnip, rutabaga, carrot, and red beet revealed that they contained only ferulic acid (0.05-0.22%). Ferulic acid was identified as a constituent of the insoluble material recovered after partial hydrolysis of potato and beet suberins (34% and 32% loss, respectively) in 1 m NaOH at 80 C. A major part (65%) of the ferulic acid contained in potato suberin was contained in the insoluble material recovered after partial (26.8% loss) degradation of this suberin with 0.01 m NaOH. Ferulic acid appears to be tightly (probably covalently) bound to suberin.     

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.     

Glycerol-derived ester oligomers from cork suberin

The cork suberin polyester was partially depolymerized by a methanolysis reaction catalyzed by calcium hydroxide. The methanolisate was analysed by ESI-MS/MS in the form of [M+Li](+) adduct-ions. This reaction solubilized a mixture of monomers and oligomers, including a set of glycerol-derived dimeric and trimeric esters. Four types of glycerol esters were identified: monoacylglycerols of alpha,omega-diacids, of omega-hydroxyacids and of monoacids; diglycerol diesters of alpha,omega-diacids; diacylglycerols of alpha,omega-diacids; monoacylglycerols of linear dimeric esters of alpha,omega-diacids and omega-hydroxyacids. The alpha,omega-diacids and omega-hydroxyacids found as monomer residues in the glycerol esters are the main ones found as cork suberin monomers. It is concluded that suberin is a glycerol-derived lipid of polymeric dimensions. Due to the protective and insulating role that it plays in plants, suberin should be considered together with the other known glycerolipids that build up biological membranes.     

Diversity of cutinases from plant pathogenic fungi: differential and sequential expression of cutinolytic esterases by Alternaria brassicicola

Plant cuticles provide a protective layer that has to be penetrated by fungal pathogens. Evidence is provided for a differential and sequential induction of two classes of cutinolytic esterases by Alternaria brassicicola. Serine esterases with cutinolytic activities were expressed by conidia germinating on host surfaces. The enzymes were not induced by surface wax or cutin monomers. They were only expressed during initial (24 h) contact of conidia with cutin on host surfaces freed from wax, and with cutin in aqueous suspensions. In contrast, contact with cutin had no immediate effect on the expression of CUTAB1, a gene encoding two cutinase isozymes with crucial functions in the saprophytic utilization of cutin. Presence of a cutin monomer or prolonged exposure to cutin was required for the induction of CUTAB1 expression. The differential induction of cutinolytic esterases indicates a sequential recognition of cutin as a barrier to be penetrated and to be utilized as a carbon source in saprophytic stages.     

Formation of plant cuticle: evidence for the occurrence of the peroxygenase pathway

Cuticle plays a major role as a protective barrier in plants. Despite its physiological importance, the mode of formation of this complex structure remains poorly understood. In particular, none of the putative enzymes involved in the biosynthesis of the cutin, the matrix of cuticle, have been cloned. We have shown previously that peroxygenase is able to catalyze in vitro the epoxidation step required for the biosynthesis of C18 cutin monomers. In the present work, we have confirmed in planta that this oxidase is indeed a key enzyme involved in the formation of cutin. Thus, in maize leaves, the specific inactivation of peroxygenase by organophosphorothioates resulted in a dramatic decrease of cuticular epoxide content, as visualized by a specific histochemical technique that was accompanied by a reduced thickness of the cuticle. A strict correlation could also be established between the extent of inhibition of the peroxygenase and the modification of the cuticle triggered by a family of structurally related inhibitors. Importantly, these effects were restricted to plants that contain a cutin originating from C18 monomers. The altered cuticle of maize, treated with the peroxygenase inhibitor, was characterized by an increased permeability to pesticides. In addition, such plants became largely susceptible to infection by fungi, implying that the cuticle represents a crucial target for the modulation of the response in plant-pathogen interactions.     

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.     

omega-Hydroxyacid derivatives in the epidermis of several mammalian species

1. omega-Hydroxyacids from the acylceramides and acylglucosylceramides of mammalian epidermis were examined by thin-layer and gas-liquid chromatography to determine their degree of unsaturation and chain-length distributions. The species examined included human (Homo sapiens), pig (Sus scrofa), mouse (Mus musculus) and rat (Rattus rattus). 2. Within a species, the omega-hydroxyacids from the acylceramide and acylglucosylceramide were essentially identical. 3. The human omega-hydroxyacids proved to be mainly saturated with C30:0 being the major entity. 4. The pig contained similar saturated, monoenoic and small amounts of dienoic omega-hydroxyacids, with C30:0, C32:1 being the major entities. 5. The mouse and rat contained C32:0 and C34:1 as the major components.     

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extracellular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that pancreatic lipase is responsible for the hydrolysis of cutin,a biopolyester present in mammalian diet,and the role of bile salt and colipase in this hydrolysis

The enzyme, which catalyzes hydrolysis of cutin, an insoluble biopolyester of hydroxy and epoxy fatty acids, was purified from porcine pancreas. With three different purification methods, previously used for the purification of pancreatic lipase, it is shown that cutin hydrolase is pancreatic lipase. This enzyme released oligomers and all types of monomers from the polymer with a pH optimum around 7.5. Taurodeoxycholate inhibited cutin hydrolysis by lipase and colipase reversed this inhibition. Evidence is presented which suggests that bile salt stabilizes the enzyme at the surface of the insoluble substrate and that the interaction of the polymer surface with the lipase-colipase-bile salt system is similar to that previously observed with triglycerides. Diethyl-p-nitrophenyl phosphate inhibited cutin hydrolysis by lipase but the hydrolysis was insensitive to diisopropyl fluorophosphate.     

The Arabidopsis DESPERADO/AtWBC11 transporter is required for cutin and wax secretion

The cuticle fulfills multiple roles in the plant life cycle, including protection from environmental stresses and the regulation of organ fusion. It is largely composed of cutin, which consists of C(16-18) fatty acids. While cutin composition and biosynthesis have been studied, the export of cutin monomers out of the epidermis has remained elusive. Here, we show that DESPERADO (AtWBC11) (abbreviated DSO), encoding a plasma membrane-localized ATP-binding cassette transporter, is required for cutin transport to the extracellular matrix. The dso mutant exhibits an array of surface defects suggesting an abnormally functioning cuticle. This was accompanied by dramatic alterations in the levels of cutin monomers. Moreover, electron microscopy revealed unusual lipidic cytoplasmatic inclusions in epidermal cells, disappearance of the cuticle in postgenital fusion areas, and altered morphology of trichomes and pavement cells. We also found that DSO is induced by salt, abscisic acid, and wounding stresses and its loss of function results in plants that are highly susceptible to salt and display reduced root branching. Thus, DSO is not only essential for developmental plasticity but also plays a vital role in stress responses.     

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.     

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.     

Perception of free cutin monomers by plant cells

Enzymatic degradation of plant cuticles by fungal pathogens results in the release of free cutin monomers. The hypothesis that free cutin monomers are recognized by plant cells as endogenous stress-related signals was tested in a model system consisting of cultured potato cells. Addition of cutin monomers in the micromolar range induced a transient alkalinization of the culture medium, similar to that observed with chitin or chitotetraose that served as positive control. The cutin monomers tested varied considerably in their potential to induce alkalinization, the most and least active compounds being cis -9,10-epoxy-18-hydroxystearic acid and palmitic acid, respectively. n ,16-dihydroxypalmitic acid ( n = 8, 9 or 10) was found to be the major component of potato leaf cuticle and was among the most active cutin monomers. 9,10-Dihydroxystearic acid, an analogue of the cutin monomer threo -9,10,18-trihydroxystearic acid, exhibited biological activity in a stereoselective manner, only the naturally occurring threo -stereoisomer inducing a rapid and strong alkalinization response. Alkalinization of the culture medium was inhibited by addition of the protein-kinase inhibitor K-252a, and the onset of alkalinization was paralleled by changes in phosphorylation of specific proteins. The active cutin monomers also stimulated the production of the plant stress hormone ethylene and activated defence-related genes at the mRNA level. The data provide evidence for a role of enzymatic breakdown products of plant cuticles as a new class of endogenous signal molecules.     

Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester

The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C(16) and C(18) unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm.     

A functional cutin matrix is required for plant protection against water loss

The plant cuticle, a cutin matrix embedded with and covered by wax, seals the aerial organ''s surface to protect the plant against uncontrolled water loss. The cutin matrix is essential for the cuticle to function as a barrier to water loss. Recently, we identified from wild barley a drought supersensitive mutant, eibi1, which is caused by a defective cutin matrix as the result of the loss of function of HvABCG31, an ABCG full transporter. Here, we report that eibi1 epidermal cells contain lipid-like droplets, which are supposed to consist of cutin monomers that have not been transported out of the cells. The eibi1 cuticle is fragile due to a defective cutin matrix. The rice ortholog of the EIBI1 gene has a similar pattern of expression, young shoot but not flag leaf blade, as the barley gene. The model of the function of Eibi1 is discussed. The HvABCG31 full transporter functions in the export of cutin components and contributed to land plant colonization, hence also to terrestrial life evolution.Key words: ABC transporter, cuticle, cuticular wax, drought resistance, inclusion