Plant material.

Fruit of sweet cherry ‘Adriana’, ‘Burlat’, ‘Hedelfinger’, ‘Merchant’, ‘Regina’, ‘Sam’, and ‘Samba’ grafted on ‘Gisela5’ (Prunus cerasus L. × Prunus canescens Bois) were collected from greenhouse-grown trees in the 2013 and 2014 seasons. All fruit were obtained from the experimental station of the Leibniz University, Hannover, Germany (lat. 52°14′N, long. 9°49′E). Water was supplied via drip irrigation. The drip irrigation was operated for 2-h intervals (corresponding to ≈15 mm of precipitation) as needed based on visual inspection of the moisture of the top soil (<20 cm). Fruit were harvested at maturity and selected for uniformity (based on color and size) and were free of visual defects. All fruit for mechanical testing were processed within 12 h of harvest. When held for more than 2 h, fruit was placed in a cold room at 5 °C, for shorter periods of time fruit was held at ambient temperature and covered with a damp paper towel. The ‘Regina’ and ‘Burlat’ fruit used for microscopy of the cross-sections of the fruit surface were fixed in Karnovsky solution (Karnovsky, 1965).

The elastometer.

For a detailed description of the elastometer, the reader is referred to Brüggenwirth et al. (2014). Briefly, an excised exocarp segment [ES (synonym fruit skin segment)] is mounted on a polymethylmethacrylate (Lucite^®; Mitsubishi Rayon Lucite Group, Southampton, UK) chamber filled with silicone oil (AK10; Wacker Chemie, Munich, Germany). The chamber is pressurized by driving a motorized piston into it, causing the volume of the chamber to decrease at a rate of ≈153 μL·min⁻¹. The volume displacement causes the chamber pressure to increase and the ES to bulge outward. The pressure inside the chamber and the extent of bulging are monitored using a pressure transducer (Typ 40PC100G; Honeywell, Morristown, NJ) and a displacement transducer (KAP-S/5N; AST Angewandte System Technik, Wolnzach, Germany) from a material testing machine (BXC-FR2.5TN; Zwick Roell, Ulm, Germany). To preserve the in vivo strain of the fruit skin following excision, a brass washer (aperture diameter 12 mm) was glued to the shoulder of an intact sweet cherry fruit using cyanoacrylate adhesive (Loctite 406; Henkel/Loctite Deutschland, Munich, Germany) (Knoche and Peschel, 2006). By cutting underneath the washer with a razor blade, an ES was excised having an average thickness of ≈2.4 ± 0.02 mm. The ES remained fixed to the washer after excision, which prevented relaxation, thereby maintaining the in vivo strain that existed before excision (Brüggenwirth et al., 2014; Knoche and Peschel, 2006). The ES so-obtained comprise cuticle, epidermis, hypodermis, and some adhering mesocarp tissue.

The surface area (A) of the bulging ES is calculated from Eq. [1], where r is the radius of the washer aperture and h is the height of the bulge ES (Brüggenwirth et al., 2014).

View Expanded

An estimate of the total strain (ɛ) is obtained from the change in surface area (ΔA) relative to the initial surface area (A₀) of the bulging ES and is given in Eq. [2].

View Expanded

In some experiments, the modulus of elasticity (E) was estimated from the height (h) of the bulging ES, the radius (r) of the aperture, the pressure (p), and the thickness (t) of the bearing layer (t = 0.1 mm) using Eq. [3] (Brüggenwirth et al., 2014).

View Expanded

All mechanical tests were performed at 22 °C.

Experiments.

The modulus of elasticity [E (megapascals)], the pressure at fracture [p_fracture (kilopascals)], and strain at fracture [ɛ_fracture (square millimeters per square millimeter)] was quantified for a range of cultivars. Biaxial tensile tests were carried out as described above.

Total, elastic, and creep strains and the effects of repeated loading, holding, and unloading cycles were measured for ‘Regina’ and ‘Burlat’. These cultivars were selected because of their contrasting cracking susceptibilities (Christensen, 1999). ES were excised and mounted as described above. Experiments were initiated by increasing the pressure during the loading phase. During the subsequent holding phase, pressure was maintained constant and then decreased in the unloading phase. The time period for a complete loading/holding/unloading cycle was ≈5 minutes. Preliminary experiments established that the test protocol had to be adapted to the cultivar tested because fracture limits differed between ‘Regina’ and ‘Burlat’. First, the pressure applied to ‘Burlat’ skin was about half of that in ‘Regina’. Second, the number of loading and unloading cycles was reduced by half for ‘Burlat’ skin, compared with that for ‘Regina’. Higher numbers of cycles resulted in fracture of the skins (M. Brüggenwirth, unpublished data). Three types of experiments were conducted. In the first experiment, the effect of increasing loading was studied by increasing the pressure stepwise in 5-kPa increments (0 to 40 kPa for ‘Regina’ and 0 to 20 kPa for ‘Burlat’). In the second experiment, pressure was held constant during each cycle (25 kPa for ‘Regina’ and 10 kPa for ‘Burlat’). The last experiment comprised pressures that decreased stepwise by 5-kPa increments (40 to 0 kPa for ‘Regina’ and 20 to 0 kPa for ‘Burlat’). Data were analyzed by partitioning the strain during each cycle into a total strain, an elastic strain, and a creep strain. The elastic strain was defined as the strain during the loading phase, and the creep strain was defined as the deformation occurring during the holding phase. Total strain represented the sum of the elastic strain and the creep strain.

The effect of eliminating cell turgor by destroying plasma membrane integrity was investigated in ‘Regina’ and ‘Burlat’. To do this, the ES were frozen for 30 minutes at −18 °C, then thawed for 30 minutes at 22 °C, and then tested in the elastometer.

Cracking and water uptake.

Cracking and water uptake relationships were established for ‘Regina’ and ‘Burlat’ sweet cherry fruit by incubation in deionized water at 22 °C. Water uptake was restricted to the fruit surface by previously sealing the stem cavity with silicone rubber (SE9186 Coating; Dow Corning, Midland, MI). Cracking was quantified in two batches of 25 fruit each, by visual inspection of fruit at 2, 4, 6, 8, 10, and 24 h for macroscopically visible cracks. The time to 50% fruit cracking (T₅₀) was calculated using a four-parameter sigmoidal regression model, fitted to a plot of the percentage of fruit cracking vs. time (Winkler et al., 2015). Fruit that had cracked was removed from the solution, and noncracked fruit was returned for further incubation. Water uptake was quantified gravimetrically after 0.75 and 1.5 h (n = 15). The water uptake required for 50% fruit cracking (WU₅₀) was calculated by multiplying the T₅₀ by the rate of water uptake.

Microscopy.

Dimensions of epidermal and hypodermal and parenchyma cells were quantified in fruit of ‘Regina’ and ‘Burlat’ fixed in Karnovsky solution (Karnovsky, 1965). Fruit were removed from the fixation solution and thoroughly washed in deionized water. Cross-sections were prepared from the shoulder region of the fruit, using parallel razor blades (spacing 3 mm), and the sections were incubated for 7 min in 0.1% (w/w) calcofluor white (fluorescent brightener 28; Sigma-Aldrich Chemie, Munich, Germany). Thereafter, cross-sections were rinsed with deionized water, positioned on a microscope slide, transferred to the stage of a fluorescence microscope (model BX-60; Olympus Europa, Hamburg, Germany), and viewed under ultraviolet light at ×400 (model BX-60, filter U-MWB 330–385 nm excitation wavelength, ≥420 nm emission wavelength; Olympus Europa). Calibrated micrographs were taken using a digital camera (DP71, Olympus Europa), and the digital images were analyzed (software Cell^P, Olympus Europa). For the cells of the epidermis, hypodermis, and outer mesocarp, the cell sizes were measured (one radial and two tangential diameters) and the distances of the cell centers from the cuticle were quantified along a line drawn perpendicular to the fruit surface.

Mass of cell walls.

Tissue cylinders (8 mm diameter) were punched using a biopsy punch (Kai Europe, Solingen, Germany) and cut to discs of 2.5 mm height using parallel razor blades. The discs comprised cuticle, epidermis and hypodermis, and the outer mesocarp. Discs were frozen in liquid N₂ and held at −20 °C. For analysis, discs were transferred into 3 mL of 80% ethanol, homogenized for 2 minutes, and thereafter heated to 80 °C for 15 minutes. Samples were then transferred into Eppendorf tubes and centrifuged at 20,800 g_n. The supernatant was removed, the pellet washed with 2 mL of 80% ethanol, and centrifuged again. This procedure was repeated twice using pure acetone. Thereafter, the pellet was dried at 70 °C and weighed. Dry mass of cell walls per unit fresh weight was calculated. The experiment was conducted with seven replicates comprising 10 discs per replicate.

Data analysis.

Data analysis was limited to segments that fractured in the center of the washer aperture. Segments fracturing at the edge of the aperture were excluded because artifacts resulting from the mounting procedure could not be excluded. Data are presented as means ± ses of means. Mean comparisons were made using Tukey’s studentized range test at P < 0.05 (packet multcomp 1.2–12, procedure glht, R 2.13.1; R Foundation for Statistical Computing, Vienna, Austria). Significance of coefficients of correlation (r) and of determination (r²) at the probability levels 0.05, 0.01, and 0.001 are indicated by *, **, and ***, respectively.

‘Regina’ and ‘Burlat’ differ in mechanical properties of their fruit skins.

The higher cracking susceptibility of ‘Burlat’ than of ‘Regina’ is consistent with published ratings (Christensen, 1999) and correlates with a weaker mechanical architecture of the fruit’s skin. First, the rates of water uptake did not differ, but cracking was faster (lower T₅₀) in ‘Burlat’ than in ‘Regina’. This resulted in a lower WU₅₀ in ‘Burlat’. Thus, cracking in ‘Burlat’ required only one-third the amount of water uptake as in ‘Regina’. Second, direct evidence for a weaker skin of ‘Burlat’ than ‘Regina’ comes from the biaxial tensile tests. The cultivar comparison established a lower value of E, and hence a low resistance to extension for ‘Burlat’ than for ‘Regina’ (Table 1). Also, the value for ‘Burlat’ was less than half than that for ‘Regina’ (Table 1). These two cultivars fell one at the upper end and the other at the lower end of the range of mechanical properties of the seven cultivars investigated here. The number of loading/holding/unloading cycles that could be applied without fracture in ‘Regina’ was twice that in ‘Burlat’ (M. Brüggenwirth, unpublished data). Finally, the slopes of the relationships between total strain and the elastic and the creep strain components vs. pressure were larger in ‘Burlat’ than in ‘Regina’. This indicates that a given strain was reached at a lower pressure in ‘Burlat’ than in ‘Regina’ (Fig. 3). While the relationship between the lower stiffness and lower fracture pressure of the cracking-susceptible ‘Burlat’ vs. the higher values of these of the less cracking–susceptible ‘Regina’ are merely correlative (i.e., they are not necessarily causal), nevertheless they are consistent with the general concept in material science that for a pressurized hollow vessel, a weaker shell increases the likelihood of failure.

Mechanical behavior of fruit skin in loading/unloading cycles.

Increasing the number of loading/unloading cycles did not change skin stiffness or fracture threshold (Fig. 3). Also, it made little difference whether the loads increased, decreased, or were maintained constant. Unlike tomato (Solanum lycopersicum L.) skin (Matas et al., 2004), there was no strain hardening in sweet cherry. In both cherry cultivars, elastic strain accounted for most of the deformation, whereas creep strain during the holding phases was low. These findings suggest diurnal oscillations in the diameter of a developing fruit (Measham et al., 2014) are unlikely to weaken the fruit skin.

Greater stiffness of ‘Regina’ skin is the result of different physical and chemical properties of the cell walls.

The tissue’s water relation affects its mechanical properties. For example, the shape of a turgid cell resists change as a result of the tangential tension in the cell wall. However, when we destroyed the plasma membrane by imposing a freeze:thaw cycle, the value of E remained five times higher and that of three times higher in ‘Regina’ than in ‘Burlat’. Hence, because the difference persists in spite of turgor loss, the different E and values of these two cultivars must be related to differences in the cell walls themselves—not to cell turgor differences. The reason for the different cell wall mechanical properties of ‘Burlat’ and ‘Regina’ is unknown, but a number of factors may be involved:

First, mass of cell walls per unit fruit mass was higher in ‘Regina’ than in ‘Burlat’. Also, microscopy indicates that the epidermis of ‘Regina’ has thicker anticlinal cell walls (2.6 ± 0.1 μm) than ‘Burlat’ (2.2 ± 0.1 μm) (C. Schumann, unpublished data). Cell wall thickness will affect the mechanical properties. Second, the epidermal cells of ‘Burlat’ had a higher mean anticlinal aspect ratio than those of ‘Regina’. This is consistent with greater strain during growth and/or with less strain relaxation upon preparation of skin sections for microscopy. Third, cell walls could differ in their compositions—differences in cell wall composition can result in differential mechanical behaviors (Chanliaud et al., 2002). For example, the mechanical properties, such as firmness, of a range of sweet cherry cultivars were related to the chemistry of their cell walls, in particular to the composition and solubility of pectin (Basanta et al., 2013; Batisse et al., 1996; Salato et al., 2013). At this stage we do not know which of these factors is the most critical. Based on earlier data from our laboratory a role of the cuticle in mechanical properties of the sweet cherry fruit skin can be excluded (Brüggenwirth et al., 2014).