Time course of change in mass of ‘Gala’ apples. Timescale in days after full bloom (DAFB).
Effect on cutin deposition of feeding 13C-labeled oleic acid to developing ‘Gala’ apples. Apples were fed for 7 d with oleic acid at concentrations ranging from 0 to 3.5 mM at three different stages of development using a polyethylene tube mounted on the fruit surface. Feeding was ended after 7 d and fruit were sample 14 d after termination of feeding (A) or at maturity (B). Insets: Incorporation of 13C label. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05. DAFB = days after full bloom.
Effects of oleic acid on the mass per unit area of the cuticular membrane (CM), the dewaxed CM (DCM), and the wax. Oleic acid was applied by spraying, by immersing, or by mounting a tube filled with oleic acid on the fruit surface. Fruit were harvested at 21 d [93 d after full bloom (DAFB)] (A) or at 79 d (151 DAFB) (B) after beginning of the application of oleic acid. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05.
Effect of spray application of oleic acid on the mass per unit area of the cuticular membrane (CM), the dewaxed CM (DCM), and the wax. Fruit were sampled at 79 (A and C) or at 151 (B and D) d after full bloom (DAFB) of ‘Gala’ apples (maturity). CM, DCM, and wax mass were quantified on the blushed (A and B) and the nonblushed surface (C and D). The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05.
Effect of spray application of oleic acid on the percentage of russeted surface area of ‘Gala’ apples. Fruit were harvested at maturity. The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different, Tukey’s studentized range test at P = 0.05.
Effect of spray application of oleic acid on stiffness (S) (A), maximum strain (εmax) (B), and maximum force (Fmax) (C) at failure of isolated cuticular membranes (CM) of ‘Gala’ apples. The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different, Tukey’s studentized range test at P = 0.05.
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Cuticular microcracks impair the barrier functions of the fruit cuticles of many fruit-crop species, including of apple (Malus ×domestica). Among other factors, microcracking depends on the balance between the rate of cuticle deposition and the rate of fruit growth. The objective of our study was to test the hypothesis that feeding the developing apple fruit with a precursor of cutin monomers increases cuticle deposition. Carbon-13–labeled oleic acid applied to the surface of ‘Gala’ apples was taken up and incorporated in the cutin fraction at all application concentrations. The rate of cuticle deposition decreased during development. Compared with the untreated controls, spray applications of oleic acid, submersion in an emulsion containing oleic acid, or feeding oleic acid using a tube mounted on the surface did not result in significant increases in the mass of the cuticular membrane (CM) or the dewaxed CM (DCM), or the wax per unit area. Similarly, spray applications of oleic acid had little effect on cuticle deposition. CM, DCM, and wax mass did increase slightly as the concentration of oleic acid increased, but this effect was only occasionally significant statistically. CM and wax mass were greater at 151 days than at 79 days after full bloom. Spray applications of the emulsion of oleic acid caused severe russeting at all concentrations. Uniaxial tensile tests showed no treatment effects on cuticle stiffness or maximum strain, but fracture force did decrease as the oleic acid concentration increased. Our results reveal that external applications of a precursor of cutin monomers or wax constituents do not lead to gravimetrically detectable increases in cuticle mass nor (most likely) to physiologically relevant increases in cuticle mass.
The leaf and fruit surfaces of most terrestrial plants are covered by a thin polymeric barrier: the cuticle (Jeffree 1996; Riederer 2006; Riederer and Schreiber 2001). Although very thin, this lipophilic barrier greatly limits the movement of gases, water, and other substances across the surface. In most fruit-crop species, an intact cuticle is essential to their attractive, shiny (fresh) appearance and also to their postharvest longevity. Cuticular failure impairs its barrier functions, leading to excessive rates of water loss (or uptake); to a dull or shriveled appearance; and, sometimes, to skin cracking. The incidence of pre- and postharvest fruit rot is also often increased.
In fruit, the formation of microscopic cracks in the cuticle (microcracks) is the first symptom of cuticular failure (Faust and Shear 1972a, 1972b; Peschel and Knoche 2005). Microcracks may be superficial (i.e., occurring just within the outer cuticle layers) or deep (i.e., running right through to the cell wall beneath). In apples, superficial microcracks are common (Curry 2009; Curry and Arey 2010; Knoche et al. 2018; Konarska 2013). In surface view, they tend to form just above the anticlinal cell walls of the subtending epidermal cells. As young fruit expand rapidly, the abutting anticlinal walls of neighboring epidermal cells partially debond and reorient from anticlinal to periclinal, so that cell shape (viewed in a transverse section) changes from portrait to landscape. This reorientation and shape-change process helps the skin to accommodate the very rapid increases in relative growth rate to which young fruit are subject (Knoche et al. 2018). In a fruit surface view, the cuticular microcracks form in a reticular pattern that aligns with the epidermal cell walls just below (Curry 2008; Knoche et al. 2018; Konarska 2013).
The superficial (nontraversing) microcracks can be repaired by the subsequent deposition of wax in the cracks (Curry 2008, 2009; Khanal and Knoche 2024; Khanal et al. 2021). This wax deposition partially restores the cuticle’s barrier properties (Khanal and Knoche 2024; Khanal et al. 2021; Knoche and Lang 2017). However, if a microcrack extends radially so as to traverse the cuticle, deposition of new wax is inadequate as a repair mechanism. Instead, the loss of the cuticular barrier properties triggers rapid cell division just beneath, and the differentiation of new cells with suberin-impregnated (waterproof) cell walls. These changes partially restore the lost barrier properties of the damaged cuticle, but give the skin a rough texture and a dull, red–brown appearance known as russeting (Faust and Shear 1972a, 1972b; Winkler et al. 2022). Skin spotting (Grimm et al. 2012), shriveling (Hasan et al. 2024), and macrocracking (Knoche and Lang 2017; Meyer 1944) may also occur.
Whether microcracks remain superficial or extend to traverse the cuticle depends on a delicate balance between the rate of radial extension of microcracks on the one hand, and the rate of cuticle deposition on the other (Knoche and Lang 2017). Young fruit are subject to especially high rates of cuticular strain. This occurs as a geometric consequence of their small initial size. The radial extension of microcracks is also increased by exposure of the fruit to surface moisture or to high aerial relative humidity. Developmental change in fruit shape results in uneven distributions of strain across the fruit surface (Knoche and Grimm 2008; Scharwies et al. 2014). Furthermore, uneven strain distributions may also arise from mechanical heterogeneity of the skin’s cellular structure associated with lenticels (Athoo et al. 2023; Brown and Considine 1982), with stomata (Knoche and Peschel 2007), and with the partial debonding of the anticlinal cell walls of the epidermis (Knoche et al. 2018). It can also result from an uneven size distribution of the underlying epidermal and hypodermal cells (Khanal et al. 2020).
A secondary factor in cuticular microcracking can be the rate of cuticular deposition. Cutin is deposited only on the inner surface of the cuticle (abutting the cell wall). Thus, the deposition of cutin not only tends to increase cuticle thickness, but also it fixes the accumulated strain in the outer layers of the cuticle laid down earlier (Khanal et al. 2014; Si et al. 2021c). In addition, wax deposition within the stretched cutin polymer network also fixes strain (Khanal et al. 2013a). Both deposition processes limit the radial extension of microcracks and thus tend to reduce the risk of the more serious consequences of microcracking, such as russeting (Khanal et al. 2021) and microbial infection (Guan et al. 2015), that follow upon the formation of microcracks that traverse the cuticle right through to the cell wall. Little is currently known about the factors controlling the rate of cuticle deposition (Si et al. 2021a).
One interesting possibility is that the cuticular membrane (CM) deposition rate might be increased by the external feeding of precursors to cutin monomers or wax constituents while apple fruit are developing. This idea seems plausible. First, apple cutin is deposited continuously throughout fruit development (Lai et al. 2016). Note, in this regard, that apple is unlike sweet cherry (Alkio et al. 2012; Peschel et al. 2007), strawberry (Straube et al. 2024), or plum (Knoche and Peschel 2007), in which cuticle deposition ceases early during fruit development. Second, previous studies have established that when fed to the surface of developing apples, oleic acid is taken up and incorporated in the cutin fraction (Si et al. 2021b, 2021c). Oleic acid is thus seen as a suitable precursor for cutin monomers in apples, because apple cutin contains ∼70% octadecanoic acid monomers (Holloway 1973; Walton and Kolattukudy 1972).
The objective of our study was to test the previous hypothesis that external applications of oleic acid to the surface of developing apples increases their deposition of cuticle.
The ‘Gala’ apple (Malus ×domestica Borkh.) was used in this study. Trees were grown at the Horticulture Research Station, Leibniz University Hannover, Ruthe, Germany (lat. 52.14N, long. 9.49E). All trees were cultivated according to current European Union regulations for integrated fruit production.
Fruit were harvested at weekly intervals up to 62 d after full bloom (DAFB) and thereafter at biweekly intervals. Pedicel and flower remnants at the calyx end were removed and the fruit were weighed using a digital balance. There were 30 individual fruit replicates.
Unlabeled oleic acid (>90% purity; Sigma-Aldrich Chemie, Steinheim, Germany) was dispersed in water using a surfactant polyoxyl-35 castor oil (Kolliphor®; Sigma-Aldrich, St. Louis, MO, USA) and ethanol (95% purity). The oleic acid, Kolliphor, and ethanol were mixed in a ratio of 81:6:13, respectively. The final emulsion was prepared by adding one part of this mixture to two parts deionized water. The emulsion was then vortexed for 5 min. Thereafter, the emulsion was brought up to the required volume and concentration by the addition of water. The composition of the formulation was adopted from a patent for a pharmaceutical formulation of Ritonavir (Alani and Ghosh 2006). The oleic acid concentrations in the emulsions differed among experiments as specified next.
Uniformly 13C-labeled oleic acid (>95% purity; Larodan AB, Solna, Sweden) was dispersed in a mixture of water, surfactant, and ethanol as described earlier and was fed to the fruit using polyethylene tubes mounted on the fruit surface (Khanal et al. 2021). Briefly, developing ‘Gala’ fruit of representative size and color and free of visual defects were selected and tagged at 58, 97, and 131 DAFB. The tip of a shortened Falcon polyethylene tube (length, 26 mm; inner diameter, 14 mm; Fisher Scientific, Schwerte, Germany) was mounted on the fruit surface using fast-curing nonphytotoxic silicone rubber (Dowsil™ SE 9186 Clear Sealant; Dow Toray, Tokyo, Japan). The 13C-labeled oleic acid emulsion at concentrations of 0, 50, 250, or 1000 mg·L–1 (equivalent to 0.2, 0.9, and 3.5 mM) were pipetted through a hole in the tip of the tube. An aliquot of 400 µL was applied per tube. Two additional treatments were applied. First, the empty formulation without oleic acid. This treatment is referred to as the concentration of 0 mg·L−1. Second, a deionized warer control without formulation and without polemic acid. The second formulation included a deionized water control without formulation and without oleic acid. Thereafter, the hole at the tip of the tube was sealed with silicone rubber to prevent evaporation and to maintain a constant concentration of oleic acid. After 7 d of feeding, the emulsion and the tube were removed, and the fruit surface was cleaned carefully using deionized water and soft tissue paper. The original footprint of the tube was marked using a permanent marker. Fruit were sampled from the tree twice: 19 d after feeding and at commercial maturity (151 DAFB).
Cuticles were isolated enzymatically (Orgell 1955). Epidermal skin segments were excised using a biopsy punch (diameter, 10 or 12 mm; Acuderm, Fort Lauderdale, FL, USA) or a cork borer (diameter, 24 mm). Russeted portions of the fruit surface were avoided. The epidermal skin segments were incubated in isolation solution containing pectinase (90 mL·L–1 Panzym Super E flüssig; Novozymes A/S, Krogshoejvej, Bagsvaerd, Denmark) and cellulase (5 mL·L–1 Cellubrix L; Novozymes A/S) prepared in 50 mM citric acid buffer (pH 4.0) at ambient temperature. NaN3 was added to the solution at a final concentration of 30 mM to suppress microbial growth. The enzyme solution was refreshed regularly until the CMs separated from the underlying tissue. The isolated CMs were cleaned with a fine aquarelle brush, rinsed five times with deionized water, and dried at 40 °C for ∼12 h. The mass of individual CM disks was quantified using a microbalance (CPA2P; Sartorius, Göttingen, Germany). Subsequently, epicuticular and cuticular wax was extracted using CHCl3 and MeOH (1:1, v/v) in a Soxhlet apparatus (Sigma-Aldrich, Inc. St.Louis, MO, USA). A minimum of five cycles (equivalent to 2.5 h) at 50 °C were run. The dewaxed CMs (DCMs) were air-dried for ∼12 h at 40 °C and the mass of the DCM disks were quantified (CPA2P; Sartorius). Wax mass per unit surface area was calculated by subtracting the mass per unit area of the DCM from the mass per unit area of the CM.
To determine the uptake and incorporation of oleic acid, the 13C content of the DCM was quantified using isotope ratio mass spectrometry and the procedure described by Si et al. (2021a, 2021b). The DCMs were analyzed because the simple partitioning of labeled oleic acid into the wax fraction of the CM would have caused artifacts. A disk (diameter, 4 mm) of DCM was recut from the center of a larger DCM disk (diameter, 10 or 12 mm). The recut DCM disk was weighed, crimped in a tin boat (4 × 4 mm; LabNeed, Nidderau, Germany), and analyzed in an Isotope Cube elemental analyzer (Elementar, Hanau, Germany) connected to an Isoprime precisION isotope ratio mass spectrometer (Isoprime-Elementar, Manchester, UK). The samples were burned at 1080 °C with an oxygen pulse. Combustion was catalyzed by cerium dioxide. The evolving CO2 was fed into an isotope ratio mass spectrometer, where a thermal conductivity detector measured standard C content (12C) and isotope C content (13C). By injecting a reference gas pulse, the C isotope ratio was calibrated online.
The isotope composition of C is specified in delta notation (as atomic percentage) and compared with the Vienna Pee Dee Belemnite. In addition, C (as atomic percentage) was referenced using International Atomic Energy Agency [(IAEA); Vienna, Austria] standards. Sucrose (IAEA-CH-6), cellulose (IAEA-CH-3), and caffeine (IAEA-600) were used as isotopic composition standards. An internal standard generated from spruce litter was used for quality control (Si et al. 2021a, 2021b).
The relative amount of tracer-derived C (RTracer) (additional C applied through feeding) to the total C pool (old C plus new C) was calculated usingwhere at % is the atomic percentage of the given 13C in the tracer (T) or 13C in the natural state [i.e., in a unlabeled control sample (C)]. The total amount of new carbon (MTracer) was calculated usingwhere Msample is the total mass of the sample used during the labeling procedure, %C is the C content of the respective sample, and msample is the molar mass of C in the sample. All percentages used in these equations were divided by 100 before calculation.
Using these equations, the amount of incorporation of 13C oleic acid in the DCM fraction was calculated.
A total of 25 uniformly flowering trees were selected. Five trees distributed randomly along a row were assigned randomly to one of the five treatments. The treatments comprised the oleic acid emulsions described earlier (0, 50, 250, or 1000 mg·L–1 oleic acid) plus the deionized water control. Solutions were applied to runoff using a backpack sprayer (∼0.75 L per tree). The emulsion in the sprayer was stirred vigorously to ensure uniform mixing. A total of 12 applications were made. Between 20 and 62 DAFB, applications were weekly; from 75 to 129 DAFB, applications were biweekly. The effects of oleic acid on cuticle deposition were quantified at 79 DAFB (after seven applications) and at 151 DAFB (after 12 applications). Cuticles were isolated and dewaxed as described earlier.
Because spraying oleic acid in preliminary experiments resulted in russeting, the effect of the concentration of oleic acid on russeting was quantified by visual assessment. An initial experiment was conducted to establish a procedure. Briefly, a sample of 30 fruit selected for a maximum range in russeting was selected, and the percentage of russeted surface area was estimated independently by five judges by visual assessment of individual fruit. The judges’ estimates were averaged and compared with measurements of the russeted surface area on the same apples conducted by image analysis. The fruit used for visual assessment were cut into eight thick sections by making seven cuts, normal to the pedicel–calyx axis using an apple cutter. The flesh was removed from each section and the skin spread on a glass plate. Unfortunately, insufficient contrast prevented automatic detection of the russeted peel portion based on color thresholds. To enhance contrast, the russeted portion of the peel was painted using black acrylic paint. Subsequently, the total peel area and the russeted peel area per fruit were quantified using image analysis (CellP; Olympus Europa, Hamburg, Germany). Plotting the russeted areas per fruit as assessed by the five judges vs. that quantified by image analysis yielded a close positive relationship, indicating that the visual assessment by the five judges was sufficiently precise to be used in routine analysis (Supplemental Fig. 1).
The potential effects of oleic acid on the mechanical properties of the cuticles were established using uniaxial tensile tests and the protocol described earlier (Khanal et al. 2013b). Briefly, parallel strips (width, 5 mm) were cut from isolated CM disks (diameter, 24 mm) using a custom-made ribbon cutter comprising two parallel-mounted razor blades. The CM strip was mounted in a paper frame prepared using masking tape (Tesa Krepp; Tesa Werk, Hamburg, Germany). The purpose of the frame was to prevent unintentional stress on the CM strip during handling and mounting. The free length of the CM strips mounted in the paper frame was 10 mm. Subsequently, frames with CM strips (specimens) were hydrated in deionized water for a minimum of 12 h and then mounted in a universal material testing machine (Z 0.5; Zwick/Roell, Ulm, Germany). One end of the specimen was mounted in an upper clamp that was affixed to a load cell (10 N; KAP-Z; Zwick/Roell) connected to a moveable crosshead. The other end of the specimen was mounted in a similar but fixed lower clamp. After mounting, the paper frames were cut open and the test was started. During the test, the CM strip was subjected to a continuously increasing tensile force until failure occurred (test speed, 1 mm·min–1). The applied force was recorded by the load cell; the stretched length of the CM strip was recorded by a displacement sensor. The force at which the CM strip fractured (maximum force; Fmax), the maximum extension of the CM strip (maximum strain; εmax), and the maximum slope of the force/strain curve (stiffness; S) of each specimen were determined using the statistical software package SAS v. 9.1.4 (SAS Institute, Cary, NC, USA). There were 15 to 20 replications per treatment.
Two to four individual fruit per tree from a minimum of 10 trees were selected, tagged, and assigned randomly to one of three methods of feeding: by tube, by spraying, or by immersion. Oleic acid was fed to the fruit as described earlier. Fruit were sampled twice: first, 21 d after feeding; second, at fruit maturity. The effects of oleic acid on cuticle deposition was assessed as described earlier.
Two to five individual fruit per tree were selected, tagged, and assigned randomly to one of five treatments over a minimum of 50 trees. The treatments were 0, 50, 250, or 1000 mg·L–1 oleic acid with a deionized water control. The 0 mg·L–1 concentration represented the empty formulation without active ingredient Approximately 250 to 300 mL of treatment solution was transferred to polyethylene bags and the bags mounted on the tree such that the fruit were fully submerged in the treatment solution. After 48 h of submersion, the bags were removed, and the fruit were rinsed with deionized water and blotted using soft tissue paper. Fruit were treated at three different stages of development—23, 68, and 104 DAFB—and sampled twice. The first sample was taken 21 d after the beginning of feeding; the second sample, at the mature stage. The effects of oleic acid on cuticle deposition were assessed as described earlier.
Unless data for individual fruit are shown (Supplemental Fig. 1), data are presented in the figures as means ± standard error. When not shown, error bars were smaller than the data symbols. Data were subjected to analysis of variance using SAS v. 9.1.4 (SAS Institute). Means were compared using Tukey’s Studentized range test.
Fruit mass increased sigmoidally with time (Fig. 1).
Citation: HortScience 60, 12; 10.21273/HORTSCI18917-25
The 13C-labeled oleic acid fed to the fruit surface was incorporated in the cutin fraction at all concentrations and at all feeding times (Fig. 2). Incorporation increased as the concentration of labeled and unlabeled oleic acid in the feeding solution increased (Fig. 2), whereas the amount of labeled oleic acid incorporated remained essentially constant and independent of concentration (Fig. 2, insets). There was more incorporation within 14 d after feeding at 53 DAFB, than at 97 or at 131 DAFB, which indicates the rate of cuticle deposition decreased during development (Fig. 2A). At the mature stage, the difference in incorporation between feeding at 53 and 97 DAFB had disappeared (Fig. 2B), but both were more effective than feeding at 131 DAFB (Fig. 2A).
Citation: HortScience 60, 12; 10.21273/HORTSCI18917-25
Exploring different methods of feeding revealed that application by spraying, submerging, or feeding using the tube mounted on the fruit surface did not result in significant increases in the mass of CM, DCM, or wax per unit area compared with the untreated control (Fig. 3). Regardless of the method of feeding, there was more CM, DCM, and wax deposited at maturity than at 72 DAFB.
Citation: HortScience 60, 12; 10.21273/HORTSCI18917-25
Spray application of oleic acid had little effect on cuticle deposition (Fig. 4). CM, DCM, and wax mass increased slightly as the concentration of oleic acid increased. This effect was only occasionally significant. CM and wax mass were greater at 151 DAFB than at 76 DAFB. There was no difference between the blushed and nonblushed surfaces of the fruit (Fig. 4).
Citation: HortScience 60, 12; 10.21273/HORTSCI18917-25
Spray application of the emulsion of oleic acid caused severe russeting at all concentrations (Fig. 5). Russeting increased as the oleic acid concentration increased. The russeting was the result of oleic acid in the emulsion, because there was no russeting when the 0 mg·L–1 formulation or deionized water were applied.
Citation: HortScience 60, 12; 10.21273/HORTSCI18917-25
Oleic acid had no significant effect on cuticle stiffness or maximum strain (Fig. 6A and 6B). However, the fracture force of the cuticle decreased as the concentration of oleic acid increased (Fig. 6C).
Citation: HortScience 60, 12; 10.21273/HORTSCI18917-25
When submerging developing fruit in the oleic acid emulsion at 23, 68, or 104 DAFB, there were no significant effects on deposition of CM, DCM, or wax regardless of the concentration used (Supplemental Fig. 2).
Our results indicate that the application of oleic acid increased cutin deposition in developing apple fruit only occasionally, and then only to a very small extent. At maturity, this small increase did not exceed an average of 6.4% for the cutin and 4.4% for the wax fraction. Comparing this change with the natural genetic variation in cutin and wax mass per unit area of a range of apple cultivars reveals that the genetic variation in cuticle deposition between apple cultivars is much larger (range in CM mass, 20.3 ± 0.3–36.2 ± 0.9 g·m–2) than the effect oleic acid had on cuticle deposition (Khanal et al. 2013b). This makes it unlikely that the small increase in cuticle deposition has any relevant effect on the barrier properties of the cuticle. The mechanical properties of the cuticle were largely unaffected by oleic acid. The decrease in maximum force of the cuticle as the oleic acid increased was most likely caused by microcracks in the cuticle. Oleic acid induced severe russeting, and russeting is always preceded by microcracking (Faust and Shear 1972a, 1972b; Khanal et al. 2021; Winkler et al. 2022).
The lack of a more significant effect of oleic acid on cuticle deposition was unexpected and deserves further comment. The oleic acid was taken up and incorporated in the cutin fraction of developing apple fruit in this and our earlier studies (Si et al. 2021c). Furthermore, incorporation in the wax fraction was also likely to have occurred, as oleic acid is also a precursor for a number of wax constituents, including alkanes, aldehydes, ketones, and primary and secondary alcohols (Li-Beisson et al. 2013). However, our experimental system does not distinguish between a simple partitioning of oleic acid into wax vs. the metabolic conversion and incorporation of label from oleic acid into wax constituents. Therefore, we did not quantify the 13C content of the wax fraction. For the cutin fraction, any oleic acid that partitioned into the cutin would have been extracted together with cuticular wax during dewaxing, leaving only bound and polymerized constituents in the cutin. Interestingly, increasing the concentration of oleic acid did not result in a proportional increase in cuticle deposition. We infer from this observation that the availability of the precursor does not limit cuticle deposition. Apparently, the natural production of precursors and monomers for further conversion and incorporation is not limiting.
Numerous steps are involved in cuticle deposition between the site of de novo synthesis of fatty acids in the plastids and the site of deposition of cutin and wax in the outer epidermal cell walls. Each of these steps is catalyzed by a specific enzyme (Li-Beisson et al. 2013; Yeats and Rose 2013). Which of these enzymes limits production and deposition of cutin and wax in developing apples is not known. As far as we are aware, a comprehensive analysis of the expressions of genes involved in cutin and wax synthesis and deposition throughout apple development is lacking. This is required if we are to identify the factors regulating cuticle deposition.
Our results show that external applications of a precursor of cutin monomers or wax constituents does not lead to gravimetrically detectable nor (probably) to physiologically meaningful increases in cuticle mass. This finding indicates that the availability of a precursor does not limit cuticle deposition in apples. Commercial products that claim to function as cuticle supplements by supplying the cuticle with precursors for cutin monomers must therefore be looked at with skepticism. Sound experimental evidence should be provided that the feeding of such precursors is an effective way of stimulating cuticle deposition in each fruit-crop species for which the claim is made.
Time course of change in mass of ‘Gala’ apples. Timescale in days after full bloom (DAFB).
Effect on cutin deposition of feeding 13C-labeled oleic acid to developing ‘Gala’ apples. Apples were fed for 7 d with oleic acid at concentrations ranging from 0 to 3.5 mM at three different stages of development using a polyethylene tube mounted on the fruit surface. Feeding was ended after 7 d and fruit were sample 14 d after termination of feeding (A) or at maturity (B). Insets: Incorporation of 13C label. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05. DAFB = days after full bloom.
Effects of oleic acid on the mass per unit area of the cuticular membrane (CM), the dewaxed CM (DCM), and the wax. Oleic acid was applied by spraying, by immersing, or by mounting a tube filled with oleic acid on the fruit surface. Fruit were harvested at 21 d [93 d after full bloom (DAFB)] (A) or at 79 d (151 DAFB) (B) after beginning of the application of oleic acid. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05.
Effect of spray application of oleic acid on the mass per unit area of the cuticular membrane (CM), the dewaxed CM (DCM), and the wax. Fruit were sampled at 79 (A and C) or at 151 (B and D) d after full bloom (DAFB) of ‘Gala’ apples (maturity). CM, DCM, and wax mass were quantified on the blushed (A and B) and the nonblushed surface (C and D). The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05.
Effect of spray application of oleic acid on the percentage of russeted surface area of ‘Gala’ apples. Fruit were harvested at maturity. The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different, Tukey’s studentized range test at P = 0.05.
Effect of spray application of oleic acid on stiffness (S) (A), maximum strain (εmax) (B), and maximum force (Fmax) (C) at failure of isolated cuticular membranes (CM) of ‘Gala’ apples. The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different, Tukey’s studentized range test at P = 0.05.
Contributor Notes
The publication of this article was funded by the Open Access Fund of Leibniz Universität Hannover.
We thank Leopold Sauheitl (Institute of Soil Science, Leibniz University Hannover, Germany) for his support in mass spectrometry. We also thank Alexander Lang and Andreas Winkler for helpful discussion and useful comments on an earlier version of this manuscript.
M.K. is the corresponding author. E-mail: moritz.knoche@obst.uni-hannover.de.
Time course of change in mass of ‘Gala’ apples. Timescale in days after full bloom (DAFB).
Effect on cutin deposition of feeding 13C-labeled oleic acid to developing ‘Gala’ apples. Apples were fed for 7 d with oleic acid at concentrations ranging from 0 to 3.5 mM at three different stages of development using a polyethylene tube mounted on the fruit surface. Feeding was ended after 7 d and fruit were sample 14 d after termination of feeding (A) or at maturity (B). Insets: Incorporation of 13C label. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05. DAFB = days after full bloom.
Effects of oleic acid on the mass per unit area of the cuticular membrane (CM), the dewaxed CM (DCM), and the wax. Oleic acid was applied by spraying, by immersing, or by mounting a tube filled with oleic acid on the fruit surface. Fruit were harvested at 21 d [93 d after full bloom (DAFB)] (A) or at 79 d (151 DAFB) (B) after beginning of the application of oleic acid. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05.
Effect of spray application of oleic acid on the mass per unit area of the cuticular membrane (CM), the dewaxed CM (DCM), and the wax. Fruit were sampled at 79 (A and C) or at 151 (B and D) d after full bloom (DAFB) of ‘Gala’ apples (maturity). CM, DCM, and wax mass were quantified on the blushed (A and B) and the nonblushed surface (C and D). The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different by Tukey’s Studentized range test at P = 0.05.
Effect of spray application of oleic acid on the percentage of russeted surface area of ‘Gala’ apples. Fruit were harvested at maturity. The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different, Tukey’s studentized range test at P = 0.05.
Effect of spray application of oleic acid on stiffness (S) (A), maximum strain (εmax) (B), and maximum force (Fmax) (C) at failure of isolated cuticular membranes (CM) of ‘Gala’ apples. The data points to the left of the origin on the x-axis represent the deionized water control without formulation and without oleic acid. Means followed by the same letter are not significantly different, Tukey’s studentized range test at P = 0.05.
