Structural Cuts Research Articles

Enhancing Cutin Extraction Efficiency from Industrially Derived Tomato Processing Residues by High-Pressure Homogenization.

This study primarily aimed to enhance the extraction of cutin from industrial tomato peel residues. Initially, the conventional extraction process was optimized using response surface methodology (RSM). Subsequently, high-pressure homogenization (HPH) was introduced to improve extraction efficiency and sustainability. The optimization process focused on determining the optimal conditions for conventional extraction via chemical hydrolysis, including temperature (100-130 °C), time (15-120 min), and NaOH concentration (1-3%). The optimized conditions, determined as 130 °C, 120 min, and 3% NaOH solution, yielded a maximum cutin extraction of 32.5%. Furthermore, the results indicated that applying HPH pre-treatment to tomato peels before alkaline hydrolysis significantly increased the cutin extraction yield, reaching 46.1%. This represents an approximately 42% increase compared to the conventional process. Importantly, HPH pre-treatment enabled cutin extraction under milder conditions using a 2% NaOH solution, reducing NaOH usage by 33%, while still achieving a substantial cutin yield of 45.6%. FT-IR analysis confirmed that cutin obtained via both conventional and HPH-assisted extraction exhibited similar chemical structures, indicating that the main chemical groups and structure of cutin remained unaltered by HPH treatment. Furthermore, cutin extracts from both conventional and HPH-assisted extraction demonstrated thermal stability up to approximately 200 °C, with less than 5% weight loss according to TGA analysis. These findings underscore the potential of HPH technology to significantly enhance cutin extraction yield from tomato peel residues while utilizing milder chemical hydrolysis conditions, thereby promoting a more sustainable and efficient cutin extraction process.

Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis.

The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development.

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.

Matrix-assisted laser desorption/ionization mass spectrometry imaging: a powerful tool for probing the molecular topology of plant cutin polymer.

The cutin polymers of different fruit cuticles (tomato, apple, nectarine) were examined using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) after in situ release of the lipid monomers by alkaline hydrolysis. The mass spectra were acquired from each coordinate with a lateral spatial resolution of approximately 100 μm. Specific monomers were released at their original location in the tissue, suggesting that post-hydrolysis diffusion can be neglected. Relative quantification of the species was achieved by introducing an internal standard, and the collection of data was subjected to non-supervised and supervised statistical treatments. The molecular images obtained showed a specific distribution of ions that could unambiguously be ascribed to cutinized and suberized regions observed at the surface of fruit cuticles, thus demonstrating that the method is able to probe some structural changes that affect hydrophobic cuticle polymers. Subsequent chemical assignment of the differentiating ions was performed, and all of these ions could be matched to cutin and suberin molecular markers. Therefore, this MALDI-MSI procedure provides a powerful tool for probing the surface heterogeneity of plant lipid polymers. This method should facilitate rapid investigation of the relationships between cuticle phenotypes and the structure of cutin within a large population of mutants.

Cutin synthesis: A slippery paradigm

Despite its biological importance, the mechanism of construction of cutin, the polymer matrix of plant cuticles, has not yet been elucidated. Recently, progress on lipid barrier formation of polymers such as cutin and suberin has been recently reviewed by Pollard et al.1 In their review the authors state that the ubiquitous cutin is the least understood of the plant extracellular polymers and that major questions about cutin structure and its macromolecular assembly remain to be resolved. At the time this paper was being published our research group has developed a new hypothesis on plant cutin synthesis.2

Cloning and Characterization of GLOSSY1, a Maize Gene Involved in Cuticle Membrane and Wax Production

The cuticle covering the aerial organs of land plants plays a protective role against several biotic and abiotic stresses and, in addition, participates in a variety of plant-insect interactions. Here, we describe the molecular cloning and characterization of the maize (Zea mays) GLOSSY1 (GL1) gene, a component of the pathway leading to cuticular wax biosynthesis in seedling leaves. The genomic and cDNA sequences we isolated differ significantly in length and in most of the coding region from those previously identified. The predicted GL1 protein includes three histidine-rich domains, the landmark of a family of membrane-bound desaturases/hydroxylases, including fatty acid-modifying enzymes. GL1 expression is not restricted to the juvenile developmental stage of the maize plant, pointing to a broader function of the gene product than anticipated on the basis of the mutant phenotype. Indeed, in addition to affecting cuticular wax biosynthesis, gl1 mutations have a pleiotropic effect on epidermis development, altering trichome size and impairing cutin structure. Of the many wax biosynthetic genes identified so far, only a few from Arabidopsis (Arabidopsis thaliana) were found to be essential for normal cutin formation. Among these is WAX2, which shares 62% identity with GL1 at the protein level. In wax2-defective plants, cutin alterations induce postgenital organ fusion. This trait is not displayed by gl1 mutants, suggesting a different role of the maize and Arabidopsis cuticle in plant development.

Insights into the structure of cutin and cutan from Agave americana leaf cuticle using HRMAS NMR spectroscopy

The structure of cutan, and a cutin/cutan mixture from the Agave americana leaf cuticle is herein described by use of the technique of one and two-dimensional high-resolution magic angle spinning (HRMAS) NMR spectroscopy, with added information from solid-state 13C cross-polarization magic angle spinning (CPMAS) and Bloch decay NMR spectroscopy. Cutin in the cutin/cutan mixture is found to contain ester functionalities, in line with previous degradative approaches. Furthermore, epoxy groups, free primary alcohols and carboxylic acid groups are also observed in cutin. A significant new finding is the evidence for α-branched fatty acids/esters in cutin. Cutan has mainly free primary hydroxyls, as well as long-chain carboxylic acids, which have been previously predicted to form ester linkages with trihydroxylated benzene units. Another significant finding in cutan is the evidence for benzenecarboxylic acids that are ester linked with fatty alcohols. The long-chain polymethylenic groups in cutan are shown to express a high degree of crystallinity similar to that observed in soil organic matter.

Glycerol and glyceryl esters of ω-hydroxyacids in cutins

Cutins from the leaves and fruits of seven plant species were depolymerized by NaOCH 3-methanolysis. The monomers that were released mostly included C16 and C18 ω-hydroxyacids with mid-chain oxygenated substitutions, namely epoxy and hydroxyl groups. Glycerol was also solubilized as a monomer in quantities that ranged from 1 to 14% of the methanolysates. Partial depolymerization of three cutins by CaO-methanolysis released the same monomers as had been obtained in the previous reaction, as well as small quantities of 1- and 2-monoacylglyceryl esters of ω-hydroxyacids. Molar proportions of glycerol permit the esterification of a significant part of the aliphatic ω-hydroxyacids, thereby possibly playing a major role in the polyester structure of cutin. Glycerol had not previously been known to form part of the cutin polymer.

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF3/CH3OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100-250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.

Aspects of cutin structure and formation [proceedings

Aspects of cutin structure and formation [proceedings

Studies on cutin-esterase II. Characteristics of cutin-esterase from Botrytis cinerea and its activity on tomato-cutin

The activity of cutin-esterase, cutinase, was detected in the mycelial homogenate of Botrytis cinerea cultured in a peptone-sucrose medium at 25°C for 7 days. The crude enzyme solution was prepared from the homogenate by centrifugation at 106,600×g, treatment with (NH4)2SO4 at 70% saturation, and dialysis against 0.01 M phosphate buffer. The optimum pH, temperature and assay duration for enzyme activity were 5.0, 25°C and 18 hr, respectively. Specific activity was 255 mµmoles/mg protein/18 hr as palmitate under optimum conditions. 83% of the activity was lost by heating the enzyme solution (pH 7.6) for 4 min at 95°C. Palmitic, stearic, oleic, 9, 10-dihydroxystearic or linoleic, dihydroxyeicosanoic and octadecanedioic acids were recognized in the enzymic hydrolysate of tomato-cutin using gas-liquid chromatography. Among these fatty acids, palmitic, oleic and octadecanedioic acids were readily liberated by the enzyme, but dihydroxyeicosanoic acid, the major component of tomato-cutin, was isolated only in small amounts. The enzyme is, therefore, an exo-type cutinase which hydrolyses minor side chains of fatty acids bound to the major structure of cutin. Cutinesterase may facilitate cuticular invasion by fungi as a result of reduction in mechanical strength of the cuticle by the enzyme

The cuticles of some Angiosperm leaves and fruits

SUMMARYThe 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.