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

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

The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function

The cuticle covers the aerial epidermis of land plants and plays a primary role in water regulation and protection from external stresses. Remarkable species diversity in the structure and composition of its components, cutin and wax, have been catalogued, but few functional or genetic correlations have emerged. Tomato (Solanum lycopersicum) is part of a complex of closely related wild species endemic to the northern Andes and the Galapagos Islands (Solanum Sect. Lycopersicon). Although sharing an ancestor <7 million years ago, these species are found in diverse environments and are subject to unique selective pressures. Furthermore, they are genetically tractable, since they can be crossed with S. lycopersicum, which has a sequenced genome. With the aim of evaluating the relationships between evolution, structure and function of the cuticle, we characterized the morphological and chemical diversity of fruit cuticles of seven species from Solanum Sect. Lycopersicon. Striking differences in cuticular architecture and quantities of cutin and waxes were observed, with the wax coverage of wild species exceeding that of S. lycopersicum by up to seven fold. Wax composition varied in the occurrence of wax esters and triterpenoid isomers. Using a Solanum habrochaites introgression line population, we mapped triterpenoid differences to a genomic region that includes two S. lycopersicum triterpene synthases. Based on known metabolic pathways for acyl wax compounds, hypotheses are discussed to explain the appearance of wax esters with atypical chain lengths. These results establish a model system for understanding the ecological and evolutionary functional genomics of plant cuticles.     

Fruit cuticle lipid composition during development in tomato ripening mutants

Recent studies suggest that fruit cuticle is an important contributing factor to tomato (Solanum lycopersicum) fruit shelf life and storability. Moreover, it has been hypothesized that variation in fruit cuticle composition may underlie differences in traits such as fruit resistance to desiccation and microbial infection. To gain a better understanding of cuticle lipid composition diversity during fruit ontogeny and to assess if there are common features that correlate with ripening, we examined developmental changes in fruit cuticle wax and cutin monomer composition of delayed‐ripening tomato fruit mutants, ripening inhibitor (rin) and non‐ripening (nor) and delayed‐ripening landrace Alcobaça. Previous reports show that fruit ripening processes such as climacteric ethylene production, cell wall degradation and color change are significantly delayed, or do not occur, in these lines. In the study presented here, however, we show that fruits from rin, nor and Alcobaça have cuticle lipid compositions that differ significantly from normal fruits of Ailsa Craig (AC) even at very early stages in fruit development, with continuing impacts throughout ripening. Moreover, rin, nor and the Alcobaça lines show quite different wax profiles from AC and each other throughout fruit development. Although cutin monomer composition differed much less than wax composition among the genotypes, all delayed‐ripening lines possessed higher relative amounts of C₁₈ monomers than AC. Together, these results reveal new genetic associations between cuticle and fruit development processes and define valuable genetic resources to further explore the importance of cuticle in fruit shelf life.     

The positional sterile (ps) mutation affects cuticular transpiration and wax biosynthesis of tomato fruits

Cuticular waxes are known to play a pivotal role in limiting transpirational water loss across primary plant surfaces. The astomatous tomato fruit is an ideal model system that permits the functional characterization of intact cuticular membranes and therefore allows direct correlation of their permeance for water with their qualitative and quantitative composition. The recessive positional sterile (ps) mutation, which occurred spontaneously in tomato (Solanum lycopersicum L.), is characterized by floral organ fusion and positional sterility. Because of a striking phenotypical similarity with the lecer6 wax mutant of tomato, which is defective in very-long-chain fatty acid elongation, ps mutant fruits were analyzed for their cuticular wax and cutin composition. We also examined their cuticular permeance for water following the developmental course of fruit ripening. Wild type and ps mutant fruits showed considerable differences in their cuticular permeance for water, while exhibiting similar quantitative wax accumulation. The ps mutant fruits showed a five- to eightfold increase in water loss per unit time and surface area when compared to the corresponding wild type fruits. The cuticular waxes of ps mutant fruits were characterized by an almost complete absence of n-alkanes and aldehydes, with a concomitant increase in triterpenoids and sterol derivatives. We also noted the occurrence of alkyl esters not present in the wild type. Quantitative and qualitative cutin monomer composition remained largely unaffected. The significant differences in the cuticular wax composition of ps mutant fruits induced a distinct increase of cuticular water permeance. The fruit wax compositional phenotype indicates the ps mutation is responsible for effectively blocking the decarbonylation pathway of wax biosynthesis in epidermal cells of tomato fruits.     

Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss

Plant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato ( Solanum lycopersicum ) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 ( cd1 ), cd2 and cd3 , the fruit cuticles of which have a dramatic (95–98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.     

Tomato GDSL1 Is Required for Cutin Deposition in the Fruit Cuticle

The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.     

Plant response to drought stress simulated by ABA application: Changes in chemical composition of cuticular waxes

Plant cuticles form the interface between epidermal plant cells and the atmosphere. The cuticle creates an effective barrier against water loss, bacterial and fungal infection and also protects plant tissue from UV radiation. It is composed of the cutin matrix and embedded soluble lipids also called waxes. Chemical composition of cuticular waxes and physiological properties of cuticles are affected by internal regulatory mechanisms and environmental conditions (e.g. drought, light, and humidity). Here, we tested the effect of drought stress simulation by the exogenous application of abscisic acid (ABA) on cuticular wax amount and composition. ABA-treated plants and control plants differed in total aboveground biomass, leaf area, stomatal density and aperture, and carbon isotope composition. They did not differ in total wax amount per area but there were peculiar differences in the abundance of particular components. ABA-treated plants contained significantly higher proportions of aliphatic components characterized by chain length larger than C₂₆, compared to control plants. This trend was consistent both between and within different functional groups of wax components. This can lead to a higher hydrophobicity of the cuticular transpiration barrier and thus decrease cuticular water loss in ABA-treated plants. At both ABA-treated and control plants alcohols with chain length C₂₄ and C₂₆ were predominant. Such a shift towards wax compounds having a higher average chain length under drought conditions can be interpreted as an adaptive response of plants towards drought stress.     

PENETRATION OF 2,4-D IN RELATION TO CUTICLE THICKNESS

Cuticle thickness was measured, either by direct microscopic examination or as weight per unit area, for the astomatous cuticle from the upper leaf surface of nine species and from tomato fruit. Thickness ranged from 1.4 μm for peach leaf cuticle to 10.8 μm for oleander leaf cuticle, and the weight from 0.19 mg/cm² to 1.26 mg/cm², respectively. Cuticles were isolated by the pectinase method and permeability to 2,4-D was determined. There was no correlation between cuticle thickness and penetration of 2,4-D, either for non-dewaxed cuticles or after chloroform extraction of waxes. Penetration of 2,4-D was increased following wax removal, but there was no correlation between wax content and the magnitude of the increase. It is suggested that cutin and wax qualitative composition are probably more important than thickness in determining relative permeability of cuticle from different plant species to 2,4-D.     

Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato

Plant epidermal cells have evolved specialist functions associated with adaptation to stress. These include the synthesis and deposition of specialized metabolites such as waxes and cutin together with flavonoids and anthocyanins, which have important roles in providing a barrier to water loss and protection against UV radiation, respectively. Characterization of the sticky peel (pe) mutant of tomato (Solanum lycopersicum) revealed several phenotypes indicative of a defect in epidermal cell function, including reduced anthocyanin accumulation, a lower density of glandular trichomes, and an associated reduction in trichome-derived terpenes. In addition, pe mutant fruit are glossy and peels have increased elasticity due to a severe reduction in cutin biosynthesis and altered wax deposition. Leaves of the pe mutant are also cutin deficient and the epicuticular waxes contain a lower proportion of long-chain alkanes. Direct measurements of transpiration, together with chlorophyll-leaching assays, indicate increased cuticular permeability of pe leaves. Genetic mapping revealed that the pe locus represents a new allele of CUTIN DEFICIENT2 (CD2), a member of the class IV homeodomain-leucine zipper gene family, previously only associated with cutin deficiency in tomato fruit. CD2 is preferentially expressed in epidermal cells of tomato stems and is a homolog of Arabidopsis (Arabidopsis thaliana) ANTHOCYANINLESS2 (ANL2). Analysis of cuticle composition in leaves of anl2 revealed that cutin accumulates to approximately 60% of the levels observed in wild-type Arabidopsis. Together, these data provide new insight into the role of CD2 and ANL2 in regulating diverse metabolic pathways and in particular, those associated with epidermal cells.     

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     

Rheological Properties of Enzymatically Isolated Tomato Fruit Cuticle

Rheological properties were determined for cuticular membranes (CMs) enzymatically isolated from mature tomato (Lycopersicon esculentum Mill. cv Pik Red) fruit. The cuticle responded as a viscoelastic polymer in stress-strain studies. Both CM and dewaxed CM expanded and became more elastic and susceptible to fracture when hydrated, suggesting that water plasticized the cuticle. Dewaxing of the CM caused similar changes in elasticity and fracturing, indicating that wax may serve as a supporting filler in the cutin matrix. Exposure of the cuticle to the surfactant Triton X-100 did not significantly affect its rheological properties.     

The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1

The cuticle coats the aerial organs of land plants and is composed of a cutin matrix embedded and overlayed with waxes. The Arabidopsis CER3 gene is important for cuticular wax biosynthesis and was reported to correspond to At5g02310 encoding an E3 ubiquitin ligase. Here, we demonstrate that CER3 is not At5g02310 and instead corresponds to WAX2/YRE/FLP1 (At5g57800), a gene of unknown function required for wax biosynthesis. CER3 protein has also been implicated in cutin production because strong cer3 alleles display organ fusions. Leaf cutin analysis of two cer3 alleles did not reveal significant differences in cutin load or composition, indicating that CER3 has no major role in leaf cutin formation.     

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.     

The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration barrier properties: effects of a deficiency in a beta-ketoacyl-coenzyme A synthase (LeCER6)

Cuticular waxes play a pivotal role in limiting transpirational water loss across the primary plant surface. The astomatous fruits of the tomato (Lycopersicon esculentum) 'MicroTom' and its lecer6 mutant, defective in a beta-ketoacyl-coenzyme A synthase, which is involved in very-long-chain fatty acid elongation, were analyzed with respect to cuticular wax load and composition. The developmental course of fruit ripening was followed. Both the 'MicroTom' wild type and lecer6 mutant showed similar patterns of quantitative wax accumulation, although exhibiting considerably different water permeances. With the exception of immature green fruits, the lecer6 mutant exhibited about 3- to 8-fold increased water loss per unit time and fruit surface area when compared to the wild type. This was not the case with immature green fruits. The differences in final cuticular barrier properties of tomato fruits in both lines were fully developed already in the mature green to early breaker stage of fruit development. When the qualitative chemical composition of fruit cuticular waxes during fruit ripening was investigated, the deficiency in a beta-ketoacyl-coenzyme A synthase in the lecer6 mutant became discernible in the stage of mature green fruits mainly by a distinct decrease in the proportion of n-alkanes of chain lengths > C(28) and a concomitant increase in cyclic triterpenoids. This shift in cuticular wax biosynthesis of the lecer6 mutant appears to be responsible for the simultaneously occurring increase of water permeance. Changes in cutin composition were also investigated as a function of developmental stage. This integrative functional approach demonstrates a direct relationship between cuticular transpiration barrier properties and distinct chemical modifications in cuticular wax composition during the course of tomato fruit development.     

植物角质层基因研究进展

角质层是形成于陆生植物表皮细胞壁外表面的脂质保水层。角质层的基本功能是保水,同时也在响应逆境胁迫、自我清洁及器官发育等方面发挥作用。角质层通常由角质和蜡质组成。角质是角质层的主要结构成分,其主要组分是聚酯。蜡质成分主要为极长链饱和脂肪酸及其衍生物。这些组分在内质网上合成后被转运到细胞表面,进一步形成完整的角质层结构。近年来通过对角质层相关突变体及相应基因的研究,人们对角质层在合成、转运、形成及调控等各个阶段都有了较为深入的认识。蜡质和角质的合成途径已在角质层相关基因功能的解释下逐渐浮出水面。有关角质层前体转运方面的研究,主要的突破在于ABCG全转运蛋白的发现和功能解析。在角质层形成的机理方面,角质层基因中的酯酶和脂酶类基因的研究有助于进一步认识这个复杂的过程。在基因调控方面,新的转录因子基因和角质层与环境之间的相互关系研究,也为已知的调控网络增加了新内容。该文综述了目前关于角质层相关基因的最新研究进展。