Intracuticular Wax Research Articles

Arabidopsis DREB26/ERF12 and its close relatives regulate cuticular wax biosynthesis under drought stress condition.

Land plants have evolved a hydrophobic cuticle on the surface of aerial organs as an adaptation to ensure survival in terrestrial environments. Cuticle is mainly composed of lipids, namely cutin and intracuticular wax, with epicuticular wax deposited on plant surface. The composition and permeability of cuticle have a large influence on its ability to protect plants against drought stress. However, the regulatory mechanisms underlying cuticular wax biosynthesis in response to drought stress have not been fully elucidated. Here, we identified three AP2/ERF transcription factors (DREB26/ERF12, ERF13 and ERF14) involved in the regulation of water permeability of the plant surface. Transmission electron microscopy revealed thicker cuticle on the leaves of DREB26-overexpressing (DREB26OX) plants, and thinner cuticle on the leaves of transgenic plants expressing SRDX repression domain-fused DREB26 (DREB26SR). Genes involved in cuticular wax formation were upregulated in DREB26OX and downregulated in DREB26SR. The levels of very-long chain (VLC) alkanes, which are a major wax component, increased in DREB26OX leaves and decreased in DREB26SR leaves. Under dehydration stress, water loss was reduced in DREB26OX and increased in DREB26SR. The erf12/13/14 triple mutant showed delayed growth, decreased leaf water content, and reduced drought-inducible VLC alkane accumulation. Taken together, our results indicate that the DREB26/ERF12 and its closed family members, ERF13 and ERF14, play an important role in cuticular wax biosynthesis in response to drought stress. The complex transcriptional cascade involved in the regulation of cuticular wax biosynthesis under drought stress conditions is discussed.

Cuticular Waxes and Cutin in Terminalia catappa Leaves from the Equatorial São Tomé and Príncipe Islands.

This study presents for the first time an analysis of the content and chemical composition of the cuticular waxes and cutin in the leaves of the widespread and important tropical species Terminalia catappa. The leaves were collected in the equatorial Atlantic islands of São Tomé and Príncipe, in the Gulf of Guinea. The epicuticular and intracuticular waxes were determined via dichloromethane extraction and their chemical composition via GC-MS analysis, and the content and monomeric composition of cutin were determined after depolymerization via methanolysis. The leaves contained an epidermal cuticular coverage of 52.8 μg cm-2 of the cuticular waxes (1.4% of mass) and 63.3 μg cm-2 (1.5% of mass) of cutin. Cuticular waxes include mainly n-alkanols and fatty acids, with a substantial proportion of terpenes in the more easily solubilized fraction, and sterols in the more embedded waxes. Cutin is mostly constituted by C16 fatty acids and dihydroxyacids, also including aromatic monomers, suggesting a largely linear macromolecular arrangement. The high proportion of triacontanol, α-amyrin, β-amyrin, germanicol, and lupeol in the easily solubilized cuticular fraction may explain the bioactive properties attributed to the T. catappa leaves via the popular medicine, which allows us to consider them as a potential source for the extraction of these compounds.

Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding β-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

13 CO2 labelling as a tool for elucidating the mechanism of cuticle development: a case of Clusia rosea.

The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. Epicuticular differed in composition from IW. The newly synthetised wax was deposited in IW first and later in EW. Both young and mature leaves synthetised IW and EW. The faster dynamics in young leaves were due to lower wax coverage, not a faster synthesis rate. Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of C. rosea. We discuss the ecophysiological significance of the new insights.

Orchard Net Covers Improve Resistance to Cherry Cracking Disorder.

Orchard net cover improves plant physiology, yield and fruit quality, pest and disease control, and anticipates fruit ripening. Moreover, this crop technology has been used to reduce natural cherry cracking (NCC). This is a serious physiological disorder that cracks the epidermis, the hypodermis, and the storage parenchyma layers of the fruit due to rainfall events near the harvest and it is related to low fruit osmotic potential and/or high fruit water permeability. This work aims to study the effect of orchard net cover on sweet cherry trees, cv. Early Bigi, in two harvesting years (2019 and 2021). The NCC, the induced cracking index (CI), and the cracking type incidence were determined. In addition, epicuticular and intra-cuticular wax content, biometric and physicochemical parameters were also evaluated. Net cover reduced the natural cracking index by 40%. High fruit weight values were observed in covered trees comparing to the control ones, with increases of 45% and 13%, in 2019 and 2021, respectively. A positive correlation was observed between CI and total soluble solids and a negative correlation between CI and wax content. Therefore, with forecasts of worsening heavy precipitation events near harvest, protecting cherry trees with nets will increase resistance to fruit cracking.

Effect of Seasonal Variation on Leaf Cuticular Waxes’ Composition in the Mediterranean Cork Oak (Quercus suber L.)

Quercus suber L. (cork oak) leaves were analyzed along one annual cycle for cuticular wax content and chemical composition. This species, well adapted to the long dry summer conditions prevailing in the Mediterranean, has a leaf life span of about one year. The cuticular wax revealed a seasonal variation with a coverage increase from the newly expanded leaves (115.7 µg/cm2 in spring) to a maximum value in fully expanded leaves (235.6 µg/cm2 after summer). Triterpenoids dominated the wax composition throughout the leaf life cycle, corresponding in young leaves to 26 µg/cm2 (22.6% of the total wax) and 116.0 µg/cm2 (49% of the total wax) in mature leaves, with lupeol constituting about 70% of this fraction. The total aliphatic compounds increased from 39 µg/cm2 (young leaves) to 71 µg/cm2 (mature leaves) and then decreased to 22 µg/cm2 and slightly increased during the remaining period. The major aliphatic compounds were fatty acids, mostly with C16 (hexadecanoic acid) and C28 (octacosanoic acid) chain lengths. Since pentacyclic triterpenoids are located almost exclusively within the cutin matrix (intracuticular wax), the increase in the cyclic-to-acyclic component ratio after summer shows an extensive deposition of intracuticular waxes in association with the establishment of mechanical and thermal stability and of water barrier properties in the mature leaf cuticle.

Leaf Cuticular Transpiration Barrier Organization in Tea Tree Under Normal Growth Conditions.

The cuticle plays a major role in restricting nonstomatal water transpiration in plants. There is therefore a long-standing interest to understand the structure and function of the plant cuticle. Although many efforts have been devoted, it remains controversial to what degree the various cuticular parameters contribute to the water transpiration barrier. In this study, eight tea germplasms were grown under normal conditions; cuticle thickness, wax coverage, and compositions were analyzed from the epicuticular waxes and the intracuticular waxes of both leaf surfaces. The cuticular transpiration rates were measured from the individual leaf surface as well as the intracuticular wax layer. Epicuticular wax resistances were also calculated from both leaf surfaces. The correlation analysis between the cuticular transpiration rates (or resistances) and various cuticle parameters was conducted. We found that the abaxial cuticular transpiration rates accounted for 64–78% of total cuticular transpiration and were the dominant factor in the variations for the total cuticular transpiration. On the adaxial surface, the major cuticular transpiration barrier was located on the intracuticular waxes; however, on the abaxial surface, the major cuticular transpiration barrier was located on the epicuticular waxes. Cuticle thickness was not a factor affecting cuticular transpiration. However, the abaxial epicuticular wax coverage was found to be significantly and positively correlated with the abaxial epicuticular resistance. Correlation analysis suggested that the very-long-chain aliphatic compounds and glycol esters play major roles in the cuticular transpiration barrier in tea trees grown under normal conditions. Our results provided novel insights about the complex structure–functional relationships in the tea cuticle.

Principal Component Analysis to Determine the Surface Properties That Influence the Self-Cleaning Action of Hydrophobic Plant Leaves.

It is well established that many leaf surfaces display self-cleaning properties. However, an understanding of how the surface properties interact is still not achieved. Consequently, 12 different leaf types were selected for analysis due to their water repellency and self-cleaning properties. The most hydrophobic surfaces demonstrated splitting of the νs CH2 and ν CH2 bands, ordered platelet-like structures, crystalline waxes, high-surface-roughness values, high-total-surface-free energy and apolar components of surface energy, and low polar and Lewis base components of surface energy. The surfaces that exhibited the least roughness and high polar and Lewis base components of surface energy had intracuticular waxes, yet they still demonstrated the self-cleaning action. Principal component analysis demonstrated that the most hydrophobic species shared common surface chemistry traits with low intra-class variability, while the less hydrophobic leaves had highly variable surface-chemistry characteristics. Despite this, we have shown through partial least squares regression that the leaf water contact angle (i.e., hydrophobicity) can be predicted using attenuated total reflectance Fourier transform infrared spectroscopy surface chemistry data with excellent ability. This is the first time that such a statistical analysis has been performed on a complex biological system. This model could be utilized to investigate and predict the water contact angles of a range of biological surfaces. An understanding of the interplay of properties is extremely important to produce optimized biomimetic surfaces.

Physical and Chemical Traits of Grape Varieties Influence Drosophila suzukii Preferences and Performance.

The cuticle-covered surface forms the interface between plant parts, including fruits, and their environment. The physical and chemical properties of fruit surfaces profoundly influence plant-frugivore interactions by shaping the susceptibility and suitability of the host for the attacker. Grapevine (Vitis vinifera, Vitaceae) serves as one of the various host plants of the spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), which is invasive in several parts of the world and can cause major crop losses. The susceptibility of wine towards this pest species differs widely among varieties. The objective of our study was to identify physical and chemical traits of the berry surface that may explain the differences in susceptibility of five grape varieties to D. suzukii. Both preferences of adult D. suzukii and offspring performance on intact versus dewaxed (epicuticular wax layer mechanically removed) grape berries were investigated in dual-choice assays. Moreover, the morphology and chemical composition of cuticular waxes and cutin of the different varieties were analyzed. Bioassays revealed that the epicuticular wax layer of most tested grape varieties influenced the preference behavior of adult flies; even less susceptible varieties became more susceptible after removal of these waxes. In contrast, neither offspring performance nor berry skin firmness were affected by the epicuticular wax layer. The wax morphology and the composition of both epi- and intracuticular waxes differed pronouncedly, especially between more and less susceptible varieties, while cutin was dominated by ω-OH-9/10-epoxy-C18 acid and the amount was comparable among varieties within sampling time. Our results highlight the underestimated role of the epicuticular surface and cuticle integrity in grape susceptibility to D. suzukii.

The Surfaces of the Ceratonia siliqua L. (Carob) Leaflet: Insights from Physics and Chemistry.

The production of superhydrophobic coatings inspired by the surface of plant leaves is a challenging goal. Such coatings hold a bright technological future in niche markets of the aeronautical, space, naval, building, automobile, and biomedical sectors. This work is focused on the adaxial (top) and abaxial (bottom) surfaces of the leaflet of the Ceratonia silique L. (carob), a high-commercial-value Mediterranean tree cultivated in many regions of the world. The adaxial and abaxial surfaces feature hydrophobic and superhydrophobic behaviors, respectively. Their chemical composition is very simple: monopalmitin ester and palmitic acid are protuberant in the epicuticular and intracuticular wax layers of the adaxial surface, respectively, whereas 1-octacosanol dominates in the abaxial wax layers. In both surfaces, epicuticular wax is organized along a randomly oriented and intricate network of nanometer-thick and micrometer-long plates, whose density and degree of interconnection are significantly higher in the abaxial surface. The measured tilting angles for the abaxial surface (12-70°) reveal unusual variable density and water adhesion of the nanostructured plate-based texture. Optical measurements demonstrate that light reflectance/absorbance of the glaucous abaxial surface is significantly higher/lower than that of the nonglaucous adaxial surface. In both surfaces, diffuse reflectance is dominant, and the absorbance is weakly dependent on the light incidence angle. We show that the highly dense nanostructured platelike texture of the epicuticular abaxial layer of the C. siliqua leaflet works as a sophisticated light and water management system: it reflects solar radiation diffusely to lower the surface temperature, and it has superhydrophobic character to keep the surface dry. Such attributes enable efficient gas exchange (photosynthesis and respiration), transpiration, and evaporation. To mimic for the first time the abaxial surface, a templation approach was adopted using poly(dimethylsiloxane) (PDMS)/poly(methylphenylsiloxane) (PMPS) positive/negative replicas and a soft polymer/siloxane negative replica produced by the sol-gel process. Because high topographical variations of the biotemplate and wax adhesion to the biohybrid film affected the replication quality, the reproduction of the wax texture via the synthesis of 1-octacosanol-grafted siloxane-based hybrid materials is proposed as a suitable route to duplicate the abaxial surface with high fidelity. The natural chemical/physical strategy adopted by the C. siliqua leaflet to face the harsh Mediterranean climate is a powerful source of bioinspiration for the development of diffuse reflecting and superhydrophobic material systems with foreseen applications as dual-functional antiglare and water-repelling coatings.

Plasticity of the Cuticular Transpiration Barrier in Response to Water Shortage and Resupply in Camellia sinensis: A Role of Cuticular Waxes.

The cuticle is regarded as a non-living tissue; it remains unknown whether the cuticle could be reversibly modified and what are the potential mechanisms. In this study, three tea germplasms (Wuniuzao, 0202-10, and 0306A) were subjected to water deprivation followed by rehydration. The epicuticular waxes and intracuticular waxes from both leaf surfaces were quantified from the mature 5th leaf. Cuticular transpiration rates were then measured from leaf drying curves, and the correlations between cuticular transpiration rates and cuticular wax coverage were analyzed. We found that the cuticular transpiration barriers were reinforced by drought and reversed by rehydration treatment; the initial weak cuticular transpiration barriers were preferentially reinforced by drought stress, while the original major cuticular transpiration barriers were either strengthened or unaltered. Correlation analysis suggests that cuticle modifications could be realized by selective deposition of specific wax compounds into individual cuticular compartments through multiple mechanisms, including in vivo wax synthesis or transport, dynamic phase separation between epicuticular waxes and the intracuticular waxes, in vitro polymerization, and retro transportation into epidermal cell wall or protoplast for further transformation. Our data suggest that modifications of a limited set of specific wax components from individual cuticular compartments are sufficient to alter cuticular transpiration barrier properties.

The chemical composition and potential role of epicuticular and intracuticular wax in four cultivars of table grapes

Abstract Plant cuticular wax is the first barrier to resist biotic and abiotic stress. However, little is known about the compositional differences between the epicuticular and intracuticular wax in grape berry. The compositional, morphological and functional features of cuticular wax in grape berries of Vitis vinifera cv. ‘Kyoho’, ‘Muscat Hamburg’, ‘Redglobe’, and ‘Zuijinxiang’ were investigated. The total and epicuticular wax of four berries were mainly composed of terpenoids, hydrocarbons, alcohols, fatty acids and esters. Oleanolic acid was the most abundant terpenoid among the four cultivars. Scanning electron microscopy revealed the crystalline flakes structure of the cuticular wax. Additionally, the removal of epicuticular wax accelerated the weight loss, browning, and softening of grape berries, indicating the plastic-wrap-like effect of the cuticular wax on postharvest quality. This study provided the theoretical basis for further application of the fruit wax or waxy analogue.

Chemical profiles of cuticular waxes on various organs of Sorghum bicolor and their antifungal activities

Sorghum bicolor is widely cultivated in arid and semi-arid areas. This paper reports the chemical profiles of cuticular waxes on adaxial and abaxial sides of common leaf, flag leaf, sheath and stem from six sorghum cultivars and the variations of leaf cuticular waxes at seedling, jointing and filling stages. Then, the bioassay of leaf and sheath wax were evaluated against Penicillium sp and Alternaria alternata. The six sorghum cultivars had similar wax profiles. In total, eight wax compounds were identified, including fatty acids, aldehydes, primary alcohols, alkanes, secondary alcohols, ketones, sterols and minor triterpenoids. Leaf wax coverage increased from 2.2 to 3.1 μg/cm2 at seedling stages to 6.5–14.0 μg/cm2 at jointing and filling stages, respectively. The relative abundance of primary alcohols decreased from 51 to 62% at seedling stage to 17–33% at jointing stage whereas alkanes increased from 5-9% to 19–33%. Leaf was dominated with alkanes (28.4%) and aldehydes (28.4%), sheath with acids (42.8%), and stem with aldehydes (80.8%). Epicuticular wax of leaf and sheath contained higher proportions of alkanes whereas the intracuticular waxes contained higher proportions of sterols. The leaf wax improved the growth of Penicillium but reduced that of A. alternaria, whereas sheath wax reduced the growth of Penicillium but unchanged A. alternaria. The detailed sorghum wax profiles improve our understanding of the physiological roles of these waxes and their diversified potential usages in industries.

A Proposed Method for Simultaneous Measurement of Cuticular Transpiration From Different Leaf Surfaces in Camellia sinensis.

The plant cuticle is the major barrier that limits unrestricted water loss and hence plays a critical role in plant drought tolerance. Due to the presence of stomata on the leaf abaxial surface, it is technically challenging to measure abaxial cuticular transpiration. Most of the existing reports were only focused on leaf astomatous adaxial surface, and few data are available regarding abaxial cuticular transpiration. Developing a method that can measure cuticular transpiration from both leaf surfaces simultaneously will improve our understanding about leaf transpiration barrier organization. Here, we developed a new method that enabled the simultaneous measurement of cuticular transpiration rates from the adaxial and abaxial surfaces. The proposed method combined multi-step leaf pretreatments including water equilibration under dark and ABA treatment to close stomata, as well as gum arabic or vaseline application to remove or seal the epicuticular wax layer. Mathematical formulas were established and used to calculate the transpiration rates of individual leaf surfaces from observed experimental data. This method facilitates the simultaneous quantification of cuticular transpiration from adaxial and abaxial leaf surfaces. By applying this method, we demonstrated that the adaxial intracuticular waxes and the abaxial epicuticular waxes constitute the major transpiration barriers in Camellia sinensis. Wax analysis indicated that adaxial intracuticular waxes had higher coverage of very long chain fatty acids, 1-alkanol esters, and glycols, which may be attributed to its higher transpiration barrier than that of the abaxial intracuticular waxes.

Cuticular Wax Composition of Wild and Cultivated Northern Berries.

The outer-most layer of plant surface, the cuticle, consists of epi- and intra-cuticular wax. It protects the plant from dehydration, extreme temperatures and UV radiation, as well as attacks from pests such as molds and bacteria. Berry cuticular waxes are studied to understand the metabolism character (factors affecting wax layer composition in different berry species) and increase the microbial resistance and shelf life of berries. The aim of this study was analysis of the surface wax composition of nine species of wild and cultivated berries from Northern Europe. Cuticular wax analysis were done using gas chromatography-mass spectrometry. A total of 59 different compounds were identified belonging to nine groups of compounds, namely, alkanes, phytosterols, alcohols, fatty acids, phenolic acids, ketones, aldehydes, esters and tocopherols. The analyzed blueberries had the highest amount of wax present on their surface (0.9 mg berry−1), triterpenoids were the main wax constituent in these berries, with up to 62% wax composition. Berry species and varieties were compared based on their surface wax composition—similarities were found between different blueberry varieties; however, other berries showed differences based on concentration and composition of cuticular wax.

Alkanes (C29 and C31)-Mediated Intracuticular Wax Accumulation Contributes to Melatonin- and ABA-Induced Drought Tolerance in Watermelon

As the outermost hydrophobic layer, cuticular waxes serve as an essential waterproof barrier to protect plants from desiccation, but the mechanism of wax accumulation still remains unclear. We analyzed the response of cuticular wax composition and deposition to drought in three different watermelon germplasms, namely, M20, M08, and J5F, which showed similar stomatal response, but high, moderate, and low tolerance to drought, respectively. Among the identified 28 compounds of cuticular waxes on leaves, more alkanes with chain lengths of C29 and C31 were induced in M20, accompanied by an increased transcript levels of CER1 (very-long-chain aldehyde decarbonylase 1), when compared to that in M08 and J5F. M20 showed higher total wax amount but fewer platelet-like wax crystals on the upper epidermis of leaves under drought, suggesting the prevalence of more intracuticular waxes embedded into the cutin matrix as fillers. These distinct responses of cuticular waxes in M20 conferred low water loss and high tolerance to drought. Melatonin and abscisic acid (ABA), which can be induced by drought, promoted the biosynthesis of alkanes (C29 and C31) but inhibited the accumulation of wax crystals under drought. Moreover, melatonin inhibited the elevation of ABA levels under mild drought but promoted the ABA accumulation under severe drought, indicating that melatonin and ABA function synergistically to regulate wax compositions to limit non-stomatal water loss under severe but not mild drought in watermelon. These findings can be exploited to improve crop tolerance to drought in arid regions.

Changes in Cuticle Components and Morphology of 'Satsuma' Mandarin (Citrus unshiu) during Ambient Storage and Their Potential Role on Penicillium digitatum Infection.

To elucidate the role of fruit cuticle in fungal infection, changes in cuticle composition and morphology of ‘Satsuma’ mandarin during ambient (at 25 °C) storage and their role in Penicillium digitatum infection were investigated. Results showed that the epicuticular wax yield increased from 1.11 μg cm−2 to 4.21 μg cm−2 during storage for 20 days and then decreased to 1.35 μg cm−2 as storage time prolonged to 40 days. Intracuticular wax content of fruits stored for 20 days showed a peak value that was 1.7-fold higher than that of fruits stored for 40 days. The contents of cutin monomers of fruits showed a decreased trend during storage, while their proportions in the cutin stayed stable. Acids were identified as the most abundant components in epicuticular wax independently of the storage time, followed by alkanes and terpenoids. Terpenoids were found as the predominant components in intracuticular wax during the whole storage, followed by alkanes and acids. The flattened platelets crystals of fruits at harvest changed into small granule-like wax ones after 10 days of storage then gradually distributed across the surface of the fruits as stored for 40 days. Results of in vitro tests showed that mycelial growth of Penicillium digitatum could be promoted by epicuticular wax and conidial germination could be inhibited by cutin at different storage stages. These results shed new light on the chemical basis for cuticle involvement in fungal infection.

Tender leaf and fully-expanded leaf exhibited distinct cuticle structure and wax lipid composition in Camellia sinensis cv Fuyun 6

The goal of the present study was to compare the structural and compositional differences of cuticle between tender leaf and fully-expanded leaf in Camellia sinensis, and provide metabolic base for the further characterization of wax biosynthesis in this economically important crop species. The tender second leaf and the fully-expanded fifth leaf from new twig were demonstrated to represent two different developmental stages, their cuticle thickness were measured by transmission electron microscopy. The thickness of the adaxial cuticle on the second and fifth leaf was 1.15 µm and 2.48 µm, respectively; the thickness of the abaxial cuticle on the second and fifth leaf was 0.47 µm and 1.05 µm, respectively. The thickness of the epicuticular wax layer from different leaf position or different sides of same leaf were similar. However, the intracuticular wax layer of the fifth leaf was much thicker than that of the second leaf. Total wax lipids were isolated from the second leaf and the fifth leaf, respectively. Gas chromatography-mass spectrometry analysis identified 51 wax constituents belonging to 13 chemical classes, including esters, glycols, terpenoids, fatty acids and their derivatives. Wax coverage on the second and fifth leaf was 4.76 µg/cm2 and 15.38 µg/cm2, respectively. Primary alcohols dominated in the tender second leaf. However, triterpenoids were the major components from the fully-expanded fifth leaf. The predominant carbon chains varied depending on chemical class. These data showed that the wax profiles of Camellia sinensis leaves are development stage dependent, suggesting distinct developmental dependent metabolic pathways and regulatory mechanisms.

Epicuticular wax on leaf cuticles does not establish the transpiration barrier, which is essentially formed by intracuticular wax

It is well established that waxes built up the barrier properties of cuticles, since their extraction in organic solvent e.g. chloroform increases diffusion of water and organic compounds by 1–2 orders of magnitude. Leaf surface waxes can be divided in epicuticular (on the surface of the cuticular membrane) and intracuticular (embedded in the cutin polymer) waxes. Until today there are only limited investigations dealing with the question to what extent epi- or intracuticular waxes contribute to the formation of the transpiration barrier. For Prunus laurocerasus previous studies have shown that epicuticular waxes do not contribute to the formation of the transpiration barrier. This approach successfully established for P. laurocerasus was applied to further species in order to check whether this finding also applies to a broader spectrum of species. Epicuticular wax was mechanically removed using collodion from the surface of either isolated cuticular membranes or intact leaf discs of ten further plant species differing in total wax amounts, wax compositions and transport properties. Scanning electron microscopy, which was performed to independently verify the successful removal of the surface waxes, indicated that two consecutive treatments with collodion were sufficient for a complete removal of epicuticular wax. The treated surfaces appeared smooth after removal. The total wax amounts removed with the two collodion treatments and the residual amount of waxes after collodion treatment were quantified by gas chromatography and mass spectrometry. This showed that epicuticular waxes essentially consisted of long-chain aliphatic molecules (e.g. alkanes, primary alcohols, fatty acids), whereas intracuticular wax was composed of both, triterpenoids and long-chain aliphatic molecules. Cuticular transpiration using combined replicates was measured before and after removal of surface wax. Results clearly indicated that two consecutive collodion treatments, or the corresponding solvent treatments (diethyl ether:ethanol) serving as control, did not increase cuticular transpiration of the ten further leaf species investigated. Our results lead to the conclusion that epicuticular wax does not contribute to the formation of the transpiration barrier of leaves.

Changes in cuticle compositions and crystal structure of ‘Bingtang’ sweet orange fruits (Citrus sinensis) during storage

ABSTRACTChanges in composition and structure of orange cuticle during storage at 4°C or 25°C for 40 days were investigated. The total epicuticular wax content of fruits increased during storage at 4°C for 30 days and then decreased as storage time prolonged to 40 days, while it increased continuously at 25°C for 40 days. The total intracuticular wax content of fruits increased to 9.70 μg cm−2 stored at 4°C for 10 days and then decreased to 6.74 μg cm−2 for 40 days. The total intracuticular wax content of fruits was decreased to 5.17 μg cm−2 stored at 25°C for 10 days and then increased to 10.06 μg cm−2 for 30 days. Fatty acids were the most abundant component of the epicuticular wax and terpenoids were restricted to the intracuticular wax. Terpenoids were restricted to the intracuticular wax, and their amounts in the fruit stored at 4°C increased continuously during the first 20 days of storage at 4°C and then decreased as storage time increased up to 40 days. Although significant changes were found in the contents of the cutin monomer, their proportions did not change significantly during storage at 4°C or 25°C. Size of wax platelets crystals wax increased during storage of fruits at 4°C up to 30 days; however, the crystals were degraded and decreased as storage prolonged to 40 days. Furthermore, the shape of the fruit cuticle surface wax crystals changed from flattened platelets to small granulelike after storage at 25°C for 20–40 days. The obtained results provide detailed information about the changes in orange cuticle occur during storage at varying temperatures, which may help in preserving the quality of citrus fruits during storage.