A comparison of the ultrastructure and composition of fruits’ cuticular wax from the wild-type ‘Newhall’ navel orange (Citrus sinensis [L.] Osbeck cv. Newhall) and its glossy mutant

Key message

The altered ultrastructure and composition of cuticular wax from ‘glossy Newhall’ (MT) fruits lead to its glossy phenotype.

Abstract

A novel mutant derived from the wild-type (WT) ‘Newhall’ navel orange (Citrus sinensis [L.] Osbeck cv. Newhall), named ‘glossy Newhall’ (MT), which produced much more glossy fruits that were easily distinguishable from the WT fruits was characterized in this report. The total wax loads of both WT and MT fruits varied considerably during the fruit development. The most abundant wax fraction of WT mature fruits was triterpenoids, followed by aldehydes, alkanes, fatty acids, primary alcohol and cholesterol. The total wax load in MT mature fruits was reduced by 44.2?% compared with WT. Except for the minor wax components of primary alcohol and cholesterol, the amounts of all major wax fractions in MT mature fruits were decreased in varying degrees. The major reduction occurred in aldehydes that decreased 96.4?% and alkanes that decreased 81.9?%, which was consistent with scanning electron micrographs of MT mature fruit surfaces that showed a severe loss of wax crystals. Hence, aldehydes and alkanes were suggested to be required for wax crystal formation in ‘Newhall’ navel orange fruits.     

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.     

Pilot testing ofKloeckera apiculata for the biological control of postharvest diseases of citrus

The efficacy of the yeastKloeckera apiculata, strain 34-9, in controlling postharvest decay of citrus fruit was evaluated in small-scale and pilot tests in commercial packinghouse. Kloeckera apiculata grew efficiently on different media and maintained its antagonistic activity against spore germination ofPenicillium italicum. In small-scale experiments with citrus fruits dipped in the yeast cell suspension, the development of decay in citrus was effectively inhibited. The yeast was compatible with a mixture of low concentration of a commonly chemical fungicide. In packinghouse tests, combining the yeast with 40 mg/kg Carbendazim (MBC) resulted a reduction in the incidence of decay to a level equal to that of the commercial treatment of 200 mg/kg MBC. The efficacy of the strain 34-9 could also be maintained under packinghouse conditions at a cell concentration of the yeast antagonist as low as 10⁶ cells/ml. No significant difference in the efficacy ofK. apiculata was found in either the drench or the spray application systems tested in citrus packinghouse. Scanning electron microscopy revealed attachment of the yeast cells to the pathogen hyphae. The high antagonistic activity of strain 34-9 against citrus blue mould may be related to its capability to compete withPenicillium italicum, for space and nutrients and /or involvement of directly antagonist of the yeast on the fungus.     

Commercial testing ofKloeckera apiculata, isolate 34-9, for biological control of postharvest diseases of citrus fruit

The efficacy of the yeastKloeckera apiculata strain 34–9 to control the natural incidence of postharvest decay of citrus fruit under laboratory and commercial conditions was evaluated. Small-scale experiments with citrus fruit dipped into the yeast cell suspension were carried out to test its inhibitory effect, and the development of decay in citrus was effectively inhibited. The yeast was compatible with a low concentration of a commonly used fungicide. In packinghouse tests, combining the yeast with 40 mg/L MBC (Carbendazim) resulted in a reduction in the incidence of decay, caused by the green and blue moulds (Penicillium digitatum andPenicillium italicum, respectively), equal to a conventional fungicide treatment of 200 mg/L MBC. In commercial packinghouse tests, the efficacy ofK. apiculata strain 34–9 could be maintained to be effective in controlling the decay of several cultivars under packing-house conditions at a cell concentration of the yeast antagonist 3×10⁸ cells/mL. In all experiments, after storage at 5 °C for 90 days,K. apiculata strain 34–9 did not alter any quality parameters of fruit.     

纽荷尔脐橙CCS基因的克隆与分析

利用RT-PCR技术从纽荷尔脐橙果实cDNA中分离出辣椒红／辣椒玉红素合成酶（Capsanthin/Capsorubin synthase,CCS）基因（Ccs）全长。该片断长为1619bp,编码503个氨基酸。BLAST比较其氨基酸同源性发现,该序列所推导的氨基酸与已知的甜橙CCS完全一致,与胡萝卜和辣椒的CCS,以及马铃薯新黄质合成酶（neoxanthin synthase,NSY）有70％的同源性。此外,与番茄和柑橘等的番茄红素环化酶也有50%~70%的同源性。半定量RT-PCR分析表明,Ccs在纽荷尔脐橙果实成熟过程中呈先上升后下降的表达趋势,期间9月份的表达量最高。     

植物角质层蜡质的化学组成研究综述

角质层是植物与外界的第一接触面,而角质层蜡质则是由位于角质层外的外层蜡质和深嵌在角质层中的内层蜡质两部分构成。植物角质层蜡质成分极其复杂,具有重要的生理功能。综述了有关植物角质层蜡质的化学组成信息,探讨了目前植物角质层蜡质化学成分研究中存在的一些问题,展望了角质层蜡质成分的研究前景。     

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.¹ A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer ''epicuticular'' layer as well as the blend consisting of the cutin biopolymer and ''intracuticular'' waxes.² Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Schäffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.^3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) ¹³C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS ¹³C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH₂O) chemical moieties.Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state ¹³C NMR reveals differences in the relative numbers of CHO and CH₂O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-μm length scale.     

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.     

Variations of cuticular wax in mulberry trees and their effects on gas exchange and post-harvest water loss

Though mulberry (Morus alba) tree shows great adaptations to various climate conditions, their leaf water status and photosynthesis are sensitive to climate changes. In the current study, seven widely planted mulberry cultivars in Chongqing, Southwest China, were selected to analyze leaf cuticular wax characteristics, gas exchange index, post-harvest leaf water status and their relationships, aiming to provide new theory in screening high resistant mulberry cultivars. Mulberry trees formed rounded cap-type idioblasts on the adaxial leaf surface. Film-like waxes and granule-type wax crystals covered leaf surfaces, varying in crystal density among cultivars. The stomatal aperture on the abaxial surface of cultivars with high wax amount was smaller than that of cultivars with low wax amount. The amount of total wax was negatively correlated with the net photosynthetic rate (P _N), transpiration rate (E) and stomatal conductance (g _s) and positively correlated with the moisture retention capacity. It suggested that both cuticular wax and stomatal factor might be involved in regulating water loss in mulberry leaves under field conditions. The variability in moisture retention capacity and cuticular wax characteristics might be important in evaluating and screening mulberry cultivars for increasing silk quality and silkworm productivity.     

Characterization of the components of plant cuticles in relation to the penetration of 2,4-D

Quantitative comparisons were made of the components of the cuticles of leaves of plantain, fat hen, dandelion, dock, chickweed and forget-me-not. Hydrocarbons and triterpenoids were prominent in the surface wax of plantain; esters and alcohols in the other surface waxes. Polar compounds predominated in the cuticular waxes. Cuticle development in plantain, dandelion and chickweed was similar, but the cutins differed in the relative proportions of hydroxy-fatty and fatty acid components. Sorption of 2,4-D by the cuticular membrane was inversely related to the amount of cuticular wax. Hydrocarbons and an aldehyde fraction isolated from surface wax most effectively reduced the penetration of water in a model system.     

Acyl-CoA desaturase ADS4.2 is involved in the formation of characteristic wax alkenes in young Arabidopsis leaves

Monounsaturated alkenes are present in the cuticular waxes of diverse plants and are thought to play important roles in their interactions with abiotic and biotic factors. Arabidopsis (Arabidopsis thaliana) leaf wax has been reported to contain alkenes; however, their biosynthesis has not been investigated to date. Here, we found that these alkenes have mainly ω-7 and ω-9 double bonds in characteristically long hydrocarbon chains ranging from C₃₃ to C₃₇. A screening of desaturase-deficient mutants showed that a single desaturase belonging to the acyl-CoA desaturase (ADS) family, previously reported as ADS4.2, was responsible for introducing double bonds en route to the wax alkenes. ADS4.2 was highly expressed in young leaves, especially in trichomes, where the alkenes are known to accumulate. The enzyme showed strong activity on acyl substrates longer than C₃₂ and ω-7 product regio-specificity when expressed in yeast (Saccharomyces cerevisiae). Its endoplasmic reticulum localization further confirmed that ADS4.2 has access to very-long-chain fatty acyl-CoA substrates. The upstream biosynthesis pathways providing substrates to ADS4.2 and the downstream reactions forming the alkene products in Arabidopsis were further clarified by alkene analysis of mutants deficient in other wax biosynthesis genes. Overall, our results show that Arabidopsis produces wax alkenes through a unique elongation–desaturation pathway, which requires the participation of ADS4.2.

Arabidopsis produces cuticular alkenes through a unique elongation–desaturation pathway requiring the acyl-CoA desaturase ADS4.2.     

Crystal structures and chemical composition of leaf surface wax depositions on the desert moss Syntrichia caninervis

The structures and composition of cuticular waxes deposited on the leaves of a typical desert moss, Syntrichia caninervis, were investigated. The wax crystals deposited on leaves shifted with leaf aging. The results of chemical analysis showed the main chemical components of the moss wax were fatty acids, alcohols and alkanes. Leaf aging increased the content of cuticular wax and the percentage of very long chain components, from 1150 μg g⁻¹ DW and 13.6% in younger leaves to 2640 μg g⁻¹ DW and 37.2% in aged leaves, respectively. Dehydration/hydration also augmented the wax content by 35.17% in juvenile leaves and by 1900% in lab-cultivated leaves after three-cycle treatments. Synthesis of hexadecanoic acid and tetracosane were predicted to be the first step of wax accumulation. The responses of cuticular waxes in crystal structure and chemical composition were recommended as a biomonitor for assessing the shift of ecological and environmental quality.     

Determination of diffusion coefficients of octadecanoic acid in isolated cuticular waxes and their relationship to cuticular water permeabilities

Transport properties of cuticular waxes from 40 different plant species were investigated by measuring desorption rates of ¹⁴C-labelled octadecanoic acid from isolated and subsequently reconstituted wax. Diffusion coefficients (D) of octadecanoic acid in reconstituted waxes, calculated from the slopes of the regression lines fitted to the linearized portions of desorption kinetics, ranged from 1.2 × 10^?19 m² s^?1 (Senecio kleinia leaf) to 2.9 × 10^?17 m² s^?1 (Malus cf. domestica fruit). Cuticular water permeabilities (cuticular transpiration) measured with intact cuticular membranes isolated from 24 different species varied from 1.7 × 10^?11 m s^?1 (Vanilla planifolia leaf) up to 2.1 × 10^?9 m s^?1 (Malus cf. domestica fruit), thus covering a range of more than 2 orders of magnitude. Cuticular water permeabilities were highly correlated with diffusion coefficients of octadecanoic acid in isolated cuticular wax of the same species. It is therefore possible to estimate cuticular barrier properties of stomatous leaf surfaces or of leaves where isolation of the cuticle is impossible by measuring D of octadecanoic acid in isolated waxes of these leaves.     

Leaf cuticular waxes are arranged in chemically and mechanically distinct layers: evidence from Prunus laurocerasus L.

The composition and spatial arrangement of cuticular waxes on the leaves of Prunus laurocerasus were investigated. In the wax mixture, the triterpenoids ursolic acid and oleanolic acid as well as alkanes, fatty acids, aldehydes, primary alcohols and alcohol acetates were identified. The surface extraction of upper and lower leaf surfaces yielded 280 mg m ^{? 2} and 830 mg m ^{? 2}, respectively. Protocols for the mechanical removal of waxes from the outermost layers of the cuticle were devised and evaluated. With the most selective of these methods, 130 mg m ^{? 2} of cuticular waxes could be removed from the adaxial surface before a sharp, physically resistant boundary was reached. Compounds thus obtained are interpreted as ‘epicuticular waxes’ with respect to their localization in a distinct layer on the surface of the cutin matrix. The epicuticular wax film can be transferred onto glass and visualized by scanning electron microscopy. Prunus laurocerasus epicuticular waxes consisted entirely of aliphatic compounds, whereas the remaining intracuticular waxes comprised 63% of triterpenoids. The ecological relevance of this layered structure for recognition by phytotrophic fungi and herbivorous insects that probe the surface composition for sign stimuli is discussed.     

Surface wax of Andreaea and Pogonatum species

The mosses Andreaea rupestris, Pogonatum aloides and P. urnigerum contain surface waxes in amounts of 0.05–0.12% dry wt. The waxes consisted of esters (C₃₈-C₅₄), primary alcohols (C₂₀-C₃₂), free fatty acids (C₁₆-C₃₀), and alkanes (C₂₁-C₃₁). Additionally, aldehydes (C₂₂-C₃₀) were major constituents in the wax of P. urnigerum. The classes and their chain length distributions in the surface waxes of these mosses are comparable to those of epicuticular waxes of higher plants.     

Differential Expression of Organic Acid Degradation-Related Genes During Fruit Development of Navel Oranges (Citrus sinensis) in Two Habitats

Organic acids as well as soluble sugars contribute highly to flavor and overall quality of citrus fruit. Citric acid level in fruit is influenced by several factors including environmental conditions. In this study, it was observed that different environments in two habitats (Ganzhou, Jiangxi; Songyang, Zhejiang) had minor effects on total soluble solids and citrus color index but had significant effects on organic acids levels, particularly on citric acid level, in fruit of “Newhall” and “SkaggsBonanza” navel oranges (Citrus sinensis). Expression of genes involved in citric acid biosynthesis and degradation (CitCS1, CitCS2, CitAco1, CitAco2, CitAco3, CitIDH1, CitIDH2, CitIDH3, CitGAD4, CitGAD5, and CitGS2) was analyzed in fruit grown in each of the two habitats. Citric acid biosynthesis-related citrate synthase genes were steadily expressed during navel orange fruit development, while degradation-related genes were differentially expressed. These findings suggested that the influence of different environments on fruit quality traits was predominant on the regulation of organic acids level, particularly on the degradation of citric acid. A cascade of CitAco3–CitIDH1–CitGS2 might be involved in citric acid degradation in response to different environments during fruit growth and development.     

Glossy mutants of maize. VIII. Accumulation of fatty aldehydes in surface waxes of gl5 maize seedlings

In corn seedlings (Zea mays L.) homozygous for the mutation gl5, the surface waxes are characteristically altered. In this mutant the main wax constituents (83.5%) are aldehydes while in the normal waxes alcohols predominate (62.7%). Moreover, in the normal waxes aldehydes and alcohols are made up mainly of the C₃₂ term (99%), whereas in gl5 waxes the principal aldehyde is still C₃₂ (90.7%) but the free alcohol composition pattern is noticeably modified. Here the predominant terms are C₂₄, C₂₆, and C₂₈, with C₃₂ representing only 16.6% of the total. The results indicate that the mutant induces a block in the synthesis of fatty alcohols while accumulating fatty aldehydes, the substrates from which the alcohols originate.