Abstract
Rain cracking of sweet cherry fruit (Prunus avium L.) is said to occur when the volume increase associated with water uptake, extends the fruit skin beyond its upper mechanical limits. Biaxial tensile tests recorded fracture strains (εfracture) in the range 0.17 to 0.22 mm2·mm−2 (equivalent to 17% to 22%). In these tests, an excised skin segment is pressurized from its inner surface and the resulting two-dimensional strain is quantified. In contrast, the skins of fruit incubated in water in classical immersion assays are fractured at εfracture values in the range 0.003 to 0.01 mm2·mm−2 (equivalent to 0.3% to 1%)—these values are one to two orders of magnitude lower than those recorded in the biaxial tensile tests. The markedly lower time to fracture (tfracture) in the biaxial tensile test may account for this discrepancy. The objective of our study was to quantify the effect of tfracture on the mechanical properties of excised fruit skins. The tfracture was varied by changing the rate of increase in pressure (prate) and hence, the rate of strain (εrate) in biaxial tensile tests. A longer tfracture resulted in a lower pressure at fracture (pfracture) and a lower εfracture indicating weaker skins. However, a 5-fold difference in εfracture remained between the biaxial tensile test of excised fruit skin and an immersion assay with intact fruit. Also, the percentage of epidermal cells fracturing along their anticlinal cell walls differed. It was highest in the immersion assay (94.1% ± 0.6%) followed by the long tfracture (75.3% ± 4.7%) and the short tfracture (57.3% ± 5.5%) in the biaxial tensile test. This indicates that the effect of water uptake on cracking extends beyond a mere increase in fruit skin strain resulting from a fruit volume increase. Instead, the much lower εfracture in the immersion assay indicates a much weaker skin—some other unidentified factor(s) are at work.
Rain cracking limits the production of sweet cherries in all areas of the world when rainfall occurs during the harvest season (Christensen, 1996). Fruit cracking is thought to be related to water uptake into the fruit. When the volume of the fruit increases, the skin is strained, and this must eventually lead to mechanical failure. On the basis of this concept, water uptake into the fruit and the mechanical properties of the skin are the most critical factors in cracking.
Only a few studies have addressed the mechanical properties of the skin (Bargel et al., 2004; Brüggenwirth and Knoche 2016a, 2016b; Brüggenwirth et al., 2014). Because of the high Poisson’s ratio of the sweet cherry fruit skin, realistic experiments require biaxial tensile tests such as were first described by Bargel et al. (2004). In this test, an excised segment of the fruit surface is pressurized from its inner side. As a result, the segment bulges and its surface area increases thereby mimicking the increase in area associated with water uptake into the fruit (Brüggenwirth et al., 2014). Compared with the original protocol, the test procedure has been refined by maintaining the in vivo strain of the fruit skin and by pressurizing the exocarp segments (ES; synonym fruit skin segments) with silicone oil (AK10; Wacker Chemie, Munich, Germany) rather than with water (Brüggenwirth and Knoche, 2016a, 2016b; Brüggenwirth et al., 2014). Using this protocol, the pfracture were of similar magnitude to the turgor pressures reported for individual cells or those determined at the whole-fruit level (Knoche et al., 2014; Schumann et al., 2014). However, the εfracture (range 0.17 to 0.22 mm2·mm−2) was consistently higher in the biaxial tensile tests than in immersion assays where fruit was immersed in water to determine its cracking susceptibility (Brüggenwirth and Knoche, 2016a). In these assays, a strain may be calculated from the water uptake required for 50% of the immersed fruit to crack. Typical (area) strain values in such immersion assays are in the order of 0.003 to 0.01 mm2·mm−2 (Brüggenwirth and Knoche, 2016a). The mechanistic basis for this difference in strain between the biaxial tensile tests and the immersions assays is unknown. One possible explanation would be the difference in the tfracture. In the immersions assays, fruit required on average at least 3 h to fracture, whereas in a biaxial tensile test, the ES fractured within ≈2 min. To our knowledge, the effect of tfracture on the mechanical properties of fruit skins is unknown.
The objective of this study was to quantify the effect of tfracture on the measured mechanical properties of excised sweet cherry fruit skins. We varied the tfracture by changing the prate in the biaxial tensile tests.
Materials and Methods
Plant material.
Off-season ‘Sweetheart’ sweet cherry fruit from New Zealand were purchased locally and the fruit were selected for freedom from visual defects. The rootstock and cultivation system were unknown. Fruit was in excellent fully mature condition with turgescent green pedicels. There were no signs of pitting or alligator skin or any symptoms of shriveling. Mature ‘Regina’ sweet cherry fruit was obtained from the horticultural research station of the Leibniz University at Ruthe (lat. 52°14′N, long. 9°49′E). Trees were grafted on ‘Gisela 5’ rootstocks (Prunus cerasus L. × Prunus canescens Bois) and grown in a green house. Fruit were harvested at commercial maturity based on color and taste. Because the elastometer experiment was conducted during a 5-d period, fruit were placed in cold storage at 2 °C for up to 5 d. Fruit processed in a given experiment all originated from the same batch.
The elastometer.
Thickness of the load-bearing layer was assumed to be 0.1 mm (Brüggenwirth and Knoche, 2016b). The experiment was conducted at 22 °C. The number of replicates was 10.
Water uptake and cracking assay.
Water uptake and cracking were quantified on fruit taken from the same batch used for the biaxial tensile tests. Uptake was restricted to the fruit surface by cutting the pedicel flush with the receptacle and by sealing the stem/fruit junction (the receptacle and the cut end of the pedicel) using epoxy glue (UHU plus schnellfest; UHU, Bühl/Baden, Germany). Water uptake was determined gravimetrically by immersing fruit in deionized water and weighing them 0, 0.75, and 1.5 h after immersion (n = 15). Before each weighing, a fruit was blotted dry using soft tissue paper. Cracking was quantified using a modified cracking assay as described by Verner and Blodgett (1931) and Christensen (1996). Two samples of 25 fruit each were immersed in deionized water, removed from solution at 2, 4, 6, 10, and 24 h after incubation and inspected for macroscopically visible cracks. For cracked fruit incubation was discontinued, noncracked fruit was reincubated.
Microscopy.
The mode of fracture was investigated in ‘Regina’ only. Fruit was subjected to a biaxial tensile test in the elastometer and to the immersion assay as described above. The prate were set at 0.001 and 1 kPa·s−1. Following fracture, skin sections were prepared by excising tissue from one side of a crack using a razor blade. The sections were transferred to microscope slides and were viewed in incident light at ×250 (Axioplan; Carl Zeiss Microscopy, Jena, Germany). The fracture mode was quantified by counting the number of epidermal cells where fracture occurred along the anticlinal cell walls and expressing this number as a percentage of the total number of epidermal cells adjacent to the crack. For each treatment one crack per fruit on a total of 10 fruit per treatment were examined.
Data analysis.
Occasionally, ES fractured at the edge of the orifice during biaxial tensile tests. Because of possible artifacts during mounting these ES were excluded from the analyses (Brüggenwirth et al., 2014). The data were subjected to analysis of variance. Percentage data were arcsine transformed before. Means were compared using Tukey’s studentized range test [P < 0.05 (packet multcomp 1.2–12, procedure glht, R 2.13.1; R Foundation for Statistical Computing, Vienna, Austria)]. Significance of the coefficient of determination (r2) at the 0.05, 0.01, and 0.001 P levels is indicated by *, **, and ***, respectively. Except for Fig. 1A where representative traces are shown, data are presented as means ± se.
Effect of time to fracture (tfracture) of the ‘Sweetheart’ sweet cherry fruit skin on the mechanical properties. The fruit skin was strained by pressurizing the segments from the inner side. (A) Representative traces of pressure vs. time relationships for different rates of increase in pressure (prate). Arrows indicate the point of fracture. Inset: relationship between tfracture and prate. (B–D) Effect of tfracture on the (B) modulus of elasticity (E), (C) pressure at fracture pfracture, and the (D) strain at fracture εfracture. Insets B to D: relationships between E, pfracture, or εfracture and tfracture redrawn on a log scale. Data in B to D represent means ± se (n = 10).
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 141, 5; 10.21273/JASHS03810-16
Results
Varying prate in the elastometer yielded linear pressure/time relationships similar to the example in Fig. 1A, where in each, the slope of the line equals the prate. The arrows indicate the pressure and time of fracture. The tfracture was inversely related to prate as indicated by the linear relationship of a log-log plot of time vs. rate [log time (h) = −0.92 (±0.014) × log rate (kPa·s−1) − 1.57 (±0.025); r2 = 0.99*** (Fig. 1A, inset)].
The stiffness of the ES as indexed by the E increased as tfracture increased (Fig. 1B). In contrast, the pfracture and εfracture decreased at longer values of tfracture indicating that failures occurred at lower pressures and lower strains when tests were carried out over more extended periods of tfracture and, hence, over lower values of prate (Fig. 1C and D). Relationships of mechanical properties with tfracture were log linear (Fig. 1B–D, insets). Hence, most changes in E, pfracture, and εfracture occurred for values of tfracture less than 3 h, with little change in these parameters thereafter.
The time course of water uptake by submerged fruit was linear, at a constant rate of uptake of 29.7 mg·h−1 (Fig. 2, inset). For an average fruit of mass 13.1 g, assuming a spherical shape and a density of 1 kg·dm−3, the cumulative water uptake of 44.6 ± 2.2 mg after 1.5 h corresponds to a strain of 0.002 mm2·mm−2. The percentage of cracked fruit increased in a sigmoidal pattern with time and with strain. The tfracture for 50% cracking was calculated at 5.17 h and the corresponding εfracture was calculated at 0.008 mm2·mm−2 (Table 1). Compared with biaxial tensile tests carried out at a similar εrate of the ES, the tfracture as measured by water uptake was only about one-third of that in the biaxial tensile tests, and the εfracture was only about one-fifth of it (Table 1).
Time course of cracking (main graph) and of water uptake (inset) in ‘Sweetheart’ sweet cherry fruit immersed in water. Dashed lines indicate times and cracking levels when 50% of the fruit were cracked. The second x axis gives the corresponding strain (ε) of the fruit skin, calculated from cumulative water uptake and fruit mass assuming the fruit to be a sphere and having a density of 1 kg·L−1.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 141, 5; 10.21273/JASHS03810-16
Comparison of mechanical properties of ‘Sweetheart’ sweet cherry fruit skin established in biaxial tensile tests at two different rates of increase in pressure and hence of strain vs. immersion assays of whole fruit. Mechanical properties were indexed by the time to fracture (tfracture) and the strain at fracture (εfracture). For the immersion assay, strain rates (εrate) were calculated from water uptake rates into the fruit. The εrate in biaxial tensile tests was measured from the increase in surface area associated with the rate of increase in pressure (prate).
Comparing low (0.001 kPa·s−1) and high prate (1 kPa·s−1) in the elastometer and data from the classical immersion assay in ‘Regina’ revealed that the mode of fracture differed between treatments. The percentage of epidermal cells that fractured along their anticlinal cell walls was highest in the immersion assay (94.1% ± 0.6%) followed by the low (75.3% ± 4.7%) and the high prate (57.3% ± 5.5%). These differences were statistically significant.
Discussion
Our study produced two new and important findings: 1) A longer tfracture results in a higher value of E (stiffer) but in lower values of pfracture and εfracture (weaker) and a higher percentage of epidermal cells fracturing along rather than across their anticlinal cell walls and 2) the εfracture in a biaxial tensile test carried out at comparable values of εrate remains markedly higher than that estimated from an immersion assay.
Effect of time to fracture in biaxial tensile tests.
The mechanical properties of the ES were clearly affected by the tfracture. Several explanations may be offered. First, most biological materials exhibit viscoelastic properties in their stress/strain relationships (Niklas, 1992). This includes fruit skins (Brüggenwirth et al., 2014; Hankinson et al., 1977) and fruit cuticles (Lopez-Casado et al., 2010; Matas et al., 2004; Petracek and Bukovac, 1995). In polymer science, viscoelasticity is described in analogical terms using rheological models comprising springs and dash pots arranged in series (Maxwell model), in parallel (Voigt–Kelvin model) or in combinations of both arrangements [Burgers model (Schmiedel, 1992)]. When subjected to a tensile force, stress/strain relationships are obtained that depend on the rate at which the force is applied. In sweet cherry fruit, epidermal and hypodermal cell layers, and not the cuticle, represent the load-bearing structure (Brüggenwirth et al., 2014). Furthermore, the plasma membrane restricts the movement of water as indicated by a decrease in skin stiffness after eliminating cell turgor (Brüggenwirth and Knoche, 2016b). Thus, mechanical properties of the fruit skin reflect those of the cell walls both from a material (composition) and a structural (cells with restricted water movement) point of view (Brüggenwirth and Knoche, 2016b). Therefore, applying the above analogy to cell walls of the sweet cherry skin, it may be speculated that the cellulose represents the spring, whereas the viscous pectins of the middle lamellae and the cellular structure (due to the restricted water movement) represent the dash pot (Vincent, 1990). Consequently, at a high εrate we would expect the cellulose to be stressed and strained, whereas at low εrate the middle lamella is strained and water movement from symplast into the apoplast is induced. This hypothesis would explain why varying tfracture resulted in different stiffness, pfracture and εfracture. In addition, this explanation would also account for the observed differences in failure mode where a higher percentage of epidermal cells failed along their anticlinal cell walls at the lower prate. That the mode of failure of tissue may be affected by other factors such as turgor is not unusual as De Belie et al. (2000) demonstrated for mechanical properties of pear parenchyma.
Second, formation of cracks may depend on time. For a short tfracture a crack may initiate rapidly at low pressure but crack extension and complete failure may require more time and thus, occurs later when the pressure is already higher. This argument would account for the effect of tfracture in the biaxial tests; i.e., the linear relationship on the log scale between mechanical properties and tfracture. Also, the difference between the biaxial tensile test and the immersion assay may be accounted for by the above argument.
Tensile test vs. immersion assay.
The calculated εfracture and tfracture (Table 1) in the immersion assay are of the same order of magnitude as those of earlier studies (Christensen, 1996; Winkler et al., 2015). However, comparison of biaxial tensile tests for long tfracture with the immersion assay, reveals that the εfracture still remains higher in the tensile test. This effect was not unique for off-season ‘Sweetheart’, but could be reproduced with freshly harvested ‘Regina’. The difference in εfracture may be explained by one or several of the following factors. First, the effect of water uptake somehow extends beyond its effect on the strain of the skin that is caused by the volume increase of the fruit. For this to happen, the water taken up directly or indirectly must somehow affect the cell walls causing the mode of fracture to change. For example, an indirect effect could be the bursting of individual epidermal cells that would result in leakage of malic acid into the cell-wall space. Malic acid, in turn, has been shown to increase the permeability of the plasma membrane and to further weaken cell walls (Winkler et al., 2015). This could result in the spreading of a defect similar to a “zipper” or to a “ladder” in a piece of knitted fabric. Second, spatial heterogeneity of water uptake may lead to localized strains that exceed the average strain calculated on the basis of whole-fruit water uptake. This could result in local εfracture values, comparable to those of the biaxial tensile tests, whereas the mean values calculated on a whole-fruit basis would be much lower. However, there is no indication that preferential sites of water uptake are involved. In the immersion assays, water uptake was restricted to the fruit surface by sealing the pedicel junction with silicone rubber (Beyer et al., 2002). Third, fruit in the immersion assay usually cracked in the stylar scar region, whereas the ES in the biaxial tensile test was excised from the shoulders of the fruit. However, there was no difference in the mechanical properties between the shoulder and the stylar scar in earlier studies making the above argument less likely (Brüggenwirth and Knoche, 2016b). Fourth, an artifact caused by silicone oil permeating the tissue and weakening the cell wall in the elastometer is unlikely. When establishing the biaxial tensile test, we pressurized cell walls using water and silicone oil and found no significant difference between the two at the standard prate. However, to avoid artefacts due to cell bursting in water during longer exposure times, silicone oil was used as a standard. Finally, it may be visualized that water uptake into epidermal cells increased cell turgor in fruit in the immersion assay. To our knowledge, there is no supporting evidence for this hypothesis in the literature. Cell pressure probe (CPP) techniques are too coarse to puncture the small epidermal cells and obtain direct measurements of turgor. In our earlier study, we found no effect of water uptake on cell turgor of mesocarp cells as determined by CPP (Knoche et al., 2014). At present, the mechanistic basis for the difference in mechanical properties is not known.
Conclusions
The data demonstrate that tfracture affects the mechanical properties and the mode of fracture recorded for the ES. Part of the difference in εfracture between the biaxial tensile tests and the immersions assays is accounted for by the difference in tfracture. However, some discrepancies remain, indicating that the volume increase caused by water uptake is not the only factor involved. Apparently, water uptake affects the ES in other ways, which lead to failure at lower εfracture than predicted based on biaxial tensile tests. In addition, a higher percentage of fracture of epidermal cells along their anticlinal cell walls is observed in the immersion assay. The mechanistic basis of both effects is unknown. Because the mechanical properties of the ES are determined primarily by the cell walls (Brüggenwirth and Knoche, 2016b), further studies should focus on the interaction between water uptake and the mechanical behavior of the cell wall.
Literature Cited
Bargel, H., Spatz, H.C., Speck, T. & Neinhuis, C. 2004 Two-dimensional tension tests in plant biomechanics: Sweet cherry fruit skin as a model system Plant Biol. 6 432 439
Beyer, M., Peschel, S., Knoche, M. & Knörgen, M. 2002 Studies on water transport through the sweet cherry fruit surface: IV. Regions of preferential uptake HortScience 37 637 641
Brüggenwirth, M., Fricke, H. & Knoche, M. 2014 Biaxial tensile tests identify epidermis and hypodermis as the main structural elements of sweet cherry skin. Ann. Bot. Plants 6:plu019
Brüggenwirth, M. & Knoche, M. 2016a Factors affecting mechanical properties of the skin of sweet cherry fruit J. Amer. Soc. Hort. Sci. 141 45 53
Brüggenwirth, M. & Knoche, M. 2016b Mechanical properties of skin of sweet cherry fruit of differing susceptibilities to cracking J. Amer. Soc. Hort. Sci. 141 162 168
Christensen, J.V. 1996 Rain-induced cracking of sweet cherries. Its causes and prevention, p. 297–327. In: A.D. Webster and N.E. Looney (eds.). Cherries. CAB Intl., Wallingford, UK
De Belie, N., Hallett, I.C., Harker, F.R. & De Baerdemaeker, J. 2000 Influence of ripening and turgor on the tensile properties of pears: A microscopic study of cellular and tissue changes J. Amer. Soc. Hort. Sci. 125 350 356
Hankinson, B., Rao, V.N.M. & Smit, C.J.B. 1977 Viscoelastic and histological properties of grape skins J. Food Sci. 42 632 635
Knoche, M., Grimm, E. & Schlegel, H.J. 2014 Mature sweet cherries have low turgor J. Amer. Soc. Hort. Sci. 139 3 12
Knoche, M. & Peschel, S. 2006 Water on the surface aggravates microscopic cracking of the sweet cherry fruit cuticle J. Amer. Soc. Hort. Sci. 131 192 200
Lopez-Casado, G., Salamanca, A. & Heredia, A. 2010 Viscoelastic nature of isolated tomato (Solanum lycopersicum) fruit cuticles: A mathematical model Physiol. Plant. 140 79 88
Matas, A.J., Cobb, E.D., Bartsch, J.A., Paolillo, D.J. & Niklas, K.J. 2004 Biomechanics and anatomy of Lycopersicum esculentum fruit peels and enzyme-treated samples Amer. J. Bot. 91 352 360
Niklas, K.J. 1992 Plant biomechanics: An engineering approach to plant form and function. Univ. Chicago Press, Chicago, IL
Petracek, P.D. & Bukovac, M.J. 1995 Rheological properties of enzymatically isolated tomato fruit cuticle Plant Physiol. 109 675 679
Schmiedel, H. 1992 Handbuch der Kunstoffprüfung. Hanser, Munich, Germany
Schumann, C., Schlegel, H.J., Grimm, E., Knoche, M. & Lang, A. 2014 Water potential and its components in developing sweet cherry J. Amer. Soc. Hort. Sci. 139 349 355
Verner, L. & Blodgett, E.C. 1931 Physiological studies of the cracking of sweet cherries. Univ. Idaho Agr. Expt Sta. Bul. No. 184
Vincent, J.F.V. 1990 Fracture properties of plants Adv. Bot. Res. 17 235 287
Winkler, A., Ossenbrink, M. & Knoche, M. 2015 Malic acid promotes cracking of sweet cherry fruit J. Amer. Soc. Hort. Sci. 140 280 287
