Formation of microcracks in the cuticular membrane (CM) of epidermal segments (ES) of apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf., ‘Golden Delicious’, ‘Braeburn’, ‘Idared’, ‘Jonagold’, and ‘Topaz’; all grafted on ‘Malling.9’ rootstocks] fruit was studied after exposure of the surface of the ES to water. Potential strain of the CM on the ES was preserved by mounting a stainless steel washer on the fruit surface using an ethyl-cyanacrylate adhesive. Subsequently, ES were excised by tangentially cutting underneath the washer. The number of microcracks in the CM was established by light microscopy before and after a 48-h incubation period in deionized water. Within 48 h, the number of microcracks rapidly increased when the outer surface of ES of ‘Golden Delicious’ apple was exposed to water, but there was essentially no increase in microcracks when exposed to the ambient atmosphere. The occurrence of microcracks depended on the region of the fruit surface and increased from the rim of the pedicel cavity to the calyx. Increasing the relative humidity (greater than 75% relative humidity at 22 °C) above the outer surface of the ES exponentially increased the number of microcracks. Water-induced microcracking was not limited to ‘Golden Delicious’, but also occurred in ‘Braeburn’, ‘Jonagold’, ‘Topaz’, and, to a markedly smaller extent, in ‘Idared’ apple. The mechanism of formation of microcracks in the CM of apple fruit and their role in fruit russeting are discussed.
Moritz Knoche and Eckhard Grimm
Martin Brüggenwirth and Moritz Knoche
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.
Eckhard Grimm and Moritz Knoche
The skin is the primary load-bearing structure in a sweet cherry fruit (Prunus avium L.). Failure of the skin in rain cracking is considered to be related to water uptake. Little is known of the skin’s water potential, its osmotic potential (ΨΠ S), and turgor. The objective here was to quantify ΨΠ S relative to the osmotic potential of the flesh (ΨΠ F). Spatial resolution was achieved by monitoring plasmolysis in epidermal cells in tissue sections, incubated in selected osmotica using a light microscope method. Decreasing the osmotic potential [ΨΠ (more negative)] of the incubation medium increased the proportion (percent) of plasmolyzed epidermal cells. The pattern of increasing plasmolysis was sigmoidal with increasing osmolyte concentration. The value of ΨΠ for 50% of cells plasmolyzed, depended to some extent on the osmolyte used. The value of ΨΠ became slightly less negative for the osmolytes tested in the order: 1) mannitol, 2) sucrose, and 3) artificial cherry juice (a solution comprising the five major osmolytes of sweet cherry juice in the appropriate proportions and concentrations). There was little difference in the value of ΨΠ at 50% plasmolysis between the cultivars Hedelfinger, Sam, and Sweetheart. In all three cultivars, the value of ΨΠ F (measured for expressed juice using an osmometer) was markedly more negative than that of ΨΠ S (measured for 50% plasmolysis). Incubating skin segments in juice from the same fruit resulted in the plasmolysis of most (85.7% to 96.4%) of the epidermal cells. As fruit development progressed from stage II [27 day after full bloom (DAFB)] to the fully mature stage III (97 DAFB), plasmolysis occurred for increasingly more negative values of ΨΠ. Moreover, the difference between the osmotic potential values recorded for the flesh ΨΠ F and for the skin ΨΠ S increased. Plasmolysis of epidermal cells was accompanied by a marked swelling of their walls. The results indicate a marked difference in the osmotic potential of flesh (ΨΠ F trended more negative) and skin cells (ΨΠ S trended less negative).
Martin Brüggenwirth and Moritz Knoche
Rain cracking of sweet cherry fruit (Prunus avium L.) may be the result of excessive water uptake and/or of mechanically weak skins. The objectives were to compare mechanical properties of the skins of two cultivars of contrasting cracking susceptibility using biaxial tensile tests. We chose ‘Regina’ as the less-susceptible and ‘Burlat’ as the more-susceptible cultivar. Cracking assays confirmed that cracking was less rapid and occurred at higher water uptake in ‘Regina’ than in ‘Burlat’. Biaxial tensile tests revealed that ‘Regina’ skin was stiffer as indexed by a higher modulus of elasticity (E) and had a higher pressure at fracture (
Moritz Knoche and Stefanie Peschel
Time courses of change in 1) fruit mass and surface area, 2) deposition of the cuticular membrane (CM), 3) strain of the CM, and 4) formation of microcracks in the CM of developing fruit of european plum (Prunus domestica L. ssp. domestica) were established. Fruit mass, fruit surface area, and CM mass per fruit increased between 50 and 133 days after full bloom (DAFB). Rates of CM deposition were higher during early stage III (50–71 DAFB) when amounts of wax and cutin per fruit increased, resulting in an increase in CM thickness from 3.1 to 5.9 g·m−2. Thereafter, cutin deposition ceased and CM thickness decreased to 4.7 g·m−2 at 133 DAFB. Percentage strain, determined on enzymatically isolated CM disks using image analysis, slightly decreased from 12.0% at 50 DAFB to 4.5% at 71 DAFB, but increased thereafter, averaging about 40% at 133 DAFB. The breakpoint in the time course of strain at 71 DAFB corresponded to the change in rate of cutin deposition. Frequency of microscopic cracks in the CM was closely related to strain of the CM across different developmental stages within a cultivar (pedicel end and cheek region) and across different cultivars at maturity. There was little change in microscopic cracking up to ≈20% strain. However, microcracks markedly increased when strain exceeded 20%. Most microcracks (91.0% ± 3.7% at 133 DAFB) were associated with stomata. These data indicate that a mismatch between surface area expansion of the growing fruit and cutin deposition caused strain and subsequent microcracking of the CM of developing plum.
Marco Beyer and Moritz Knoche
Rain-induced cracking of sweet cherry (Prunus avium L.) fruit is thought to be related to water absorption through the fruit surface. Conductance for water uptake (gtot. uptake) through the fruit surface of `Sam' sweet cherry was studied gravimetrically by monitoring water penetration from a donor solution of deionized water through segments of the outer pericarp into a polyethyleneglycol (PEG) containing receiver solution. Segments consisting of cuticle plus five to eight cell layers of epidermal and hypodermal tissue were mounted in stainless steel diffusion cells. Conductance was calculated from flow rates of water across the segment and the difference in osmotic potential between donor and receiver solution. Flow rates were constant up to 12 hours and decreased thereafter. A log normal distribution of gtot. uptake was observed with a median of 0.97 × 10-7 m·s-1. Further, gtot. uptake was not affected by storage duration (up to 71 days) of fruit used as a source of segments, thickness of segments (range 0.1 to 4.8 mm), or segment area exposed in the diffusion cell. Osmolality of the receiver solution in the range from 1140 to 3400 mmol·kg-1 had no effect on gtot. uptake (1.45 ± 0.42 × 10-7 m·s-1), but gtot. uptake increased by 301% (4.37 ± 0.46 × 10-7 m·s-1) at 300 mmol·kg-1. gtot. uptake was highest in the stylar scar region of the fruit (1.44 ± 0.16 × 10-7 m·s-1) followed by cheek (1.02 ±0.21 × 10-7 m·s-1), suture (0.57 ±0.17 × 10-7 m·s-1) and pedicel cavity regions (0.22 ±0.09 × 10-7 m·s-1). Across regions, gtot. uptake was related positively to stomatal density. Extracting total cuticular wax by dipping fruit in chloroform/methanol increased gtot. uptake from 1.18 ± 0.23 × 10-7 m·s-1 to 2.58 ± 0.41 × 10-7 m·s-1, but removing epicuticular wax by cellulose acetate stripping had no effect (1.59 ± 0.28 × 10-7 m·s-1). Water flux increased with increasing temperature (range 20 to 45 °C). Conductance differed between cultivars with `Hedelfinger' sweet cherry having the highest gtot. uptake (2.81 ± 0.26 × 10-7 m·s-1), followed by `Namare' (2.68 ± 0.26 × 10-7 m·s-1), `Kordia' (0.96 ± 0.14 × 10-7 m·s-1), `Sam' (0.87 ± 0.15 × 10-7 m·s-1), and `Adriana' (0.33 ± 0.02 × 10-7 m·s-1). The diffusion cell system described herein may be useful in analyzing conductance in water uptake through the fruit surface of sweet cherry and its potential relevance for fruit cracking.
Stefanie Peschel and Moritz Knoche
Frequency and distribution of microcracks in the cuticular membrane (CM) were monitored in cheek, suture, pedicel cavity and stylar regions of developing sweet cherry (Prunus avium L.) fruit using fluorescence microscopy following infiltration with a fluorescence tracer (1 to 2 min in 0.1% w/v acridine orange containing 50 mm citric acid and 0.1% Silwet L-77, pH 6.5). These microcracks were limited to the cuticle, did not extend into the pericarp and were only detected by microscopy. Fruit mass and surface area increased in a sigmoidal pattern with time between 16 days after full bloom (DAFB) and maturity. The increase in frequency of fruit with microcracks paralleled the increase in fruit mass. During early development (up to 43 DAFB) the CM of `Sam' fruit remained intact. However, by 57 DAFB essentially all `Sam' fruit had microcracks in the pedicel cavity and ≈25% in the suture region with little change thereafter. At maturity percentage of `Sam' fruit with microcracks in cheek, suture, pedicel cavity and stylar end region averaged 23%, 25%, 100%, and 63%, respectively. Similar data were obtained for `Hedelfinger' (70% and 100% for cheek and pedicel cavity, respectively), `Kordia' (80% and 100%) and `Van' (100% and 100%). Generally, microcracks were most severe in pedicel cavity and stylar end region. Most of the first detectable microcracks formed above periclinal walls of epidermal cells perpendicular to their longest axis (72% and 92% in cheek and stylar regions, respectively). The other microcracks formed above the anticlinal walls were mostly oriented in the direction of the underlying cell wall. There was no difference in projected surface area, length/width ratio or orientation among epidermal cells below, adjacent to or distant from the first detectable microcracks in the CM. However, as length of microcracks increased the projected surface area of cells underlying cracks increased suggesting strain induced upon cracking of the CM. Permeability of excised exocarp segments in osmotic water uptake was positively correlated with number of stomata and number of microcracks in the CM. From our results we suggest that strain of the epidermal system during stage III of fruit growth is a factor in “microcracking” of the CM that may predispose fruit to subsequent rain-induced cracking.
Moritz Knoche and Stefanie Peschel
The effect of surface water on the frequency of microcracks in the cuticular membrane (CM) of exocarp segments (ES) of developing sweet cherry fruit (Prunus avium L.) was studied. Strain of CM and ES on the fruit surface was preserved by mounting a stainless steel washer on the fruit surface in the cheek region using an ethyl-cyanacrylate adhesive. ES were excised by tangentially cutting underneath the washer. Frequency of microcracks in the CM of ES was determined following infiltration for 10 minutes with a 0.1% acridine orange solution by fluorescence microscopy before and after exposure to deionized water (generally 48 hours). Exposing the surface of ES of mature `Burlat' sweet cherry fruit to water resulted in a rapid increase in microcracks in the CM that approached an asymptote at about 30 microcracks/cm2 within 24 hours. There was no change in microcracks in the CM when the surface of the ES remained dry. Incubating ES in polyethylene glycol solution that was isotonic to fruit juice extracted from the same batch of fruit resulted in a greater increase in frequency of microcracks as compared to incubation in deionized water. The water-induced increase in microcracks was closely related to strain of the CM across different developmental stages within a cultivar [between 45 and 94 days after full bloom (DAFB); r 2 = 0.96, P ≤ 0.001, n = 9] or across different cultivars at maturity (r 2 = 0.92, P ≤ 0.0022, n = 6). Incubating ES of developing fruit in enzyme solution containing pectinase and cellulase such that the outer surface remained dry resulted in complete rupture and failure of the ES. Time to rupture and percentage of ruptured ES were closely related to the strain of the CM (r 2 = 0.92, P ≤ 0.001, n = 9 and r 2 = 0.68, P ≤ 0.0063, n = 9, respectively). Removal of epicuticular wax had no effect on frequency of water-induced microcracks. Also, temperature had no effect on frequency of water-induced microcracks, but frequency of microcracks increased exponentially when exposing the outer surface of ES to relative humidities above 75%. At 100% humidity the increase in frequency of microcracks did not differ from that induced by liquid water. Local wetting the surface of intact fruit in the pedicel cavity or stylar end region resulted in formation of macroscopically visible cracks despite of a net water loss of fruit. Uniaxiale tensile tests using dry and fully hydrated CM strips isolated from mature `Sam' sweet cherry fruit established that hydration increased fracture strain, but decreased fracture stress and moduli of elasticity. Our data demonstrate that exposure of the fruit surface to liquid water or high concentrations of water vapor resulted in formation of microcracks in the CM.
Martin Brüggenwirth and Moritz Knoche
The skins of all fruit types are subject to sustained biaxial strain during the entire period of their growth. In sweet cherry (Prunus avium L.), failure of the skin greatly affects fruit quality. Mechanical properties were determined using a biaxial bulging test. The factors considered were the following: ripening, fruit water relations (including turgor, transpiration, and water uptake), and temperature. Excised discs of fruit skin were mounted in a custom elastometer and pressurized from their anatomically inner surfaces. This caused the skin disc to bulge outwards, stretching it biaxially, and increasing its surface area. Pressure (p) and biaxial strain (ε) due to bulging were quantified and the modulus of elasticity [E (synonyms elastic modulus, Young’s modulus)] was calculated. In a typical test, ε increased linearly with p until the skin fractured at p fracture and εfracture. Stiffness of the skin decreased in ripening late stage III fruit as indicated by a decrease in E. The value of p fracture also decreased, whereas that of εfracture remained about constant. Destroying cell turgor decreased E and p fracture relative to the turgescent control. The E value also decreased with increasing transpiration, while p fracture and (especially) εfracture increased. Water uptake had little effect on E, whereas εfracture and p fracture decreased slightly. Increasing temperature decreased E and p fracture, but had no effect on εfracture. Only the instantaneous elastic strain and the creep strain increased significantly at the highest temperatures. A decrease in E indicates decreasing skin stiffness that is probably the result of enzymatic softening of the cell walls of the skin in the ripening fruit, of relaxation of the cell walls on eliminating or decreasing turgor by transpiration and, possibly, of a decreasing viscosity of the pectin middle lamellae at higher temperatures. The effects are consistent with the conclusion that the epidermal and hypodermal cell layers represent the structural “backbone” of the sweet cherry fruit skin.
Stefanie Peschel and Moritz Knoche
The cuticular membrane (CM) represents the primary barrier to water uptake into sweet cherry (Prunus avium L.) fruit and thus has a central role in rain-induced cracking. The objective was to quantify CM properties potentially relevant to cracking and to estimate variance components and broad-sense heritabilities for these traits in selected sweet cherry cultivars. Within the scion cultivars, CM mass per area ranged from 0.85 g·m−2 in ‘Rainier’ to 1.61 g·m−2 in ‘Kordia’. Wax mass accounted for one-fourth of CM mass and ranged from 0.21 g·m−2 in ‘Burlat’ to 0.42 g·m−2 in ‘Zeppelin’. Biaxial elastic strain of the CM averaged 76.7% across cultivars and ranged from 56.6% in ‘Namosa’ to 97.0% in ‘Oktavia’. Strain was a linear function of fruit mass (r 2 = 0.33, P < 0.0001). Partitioning total variance into variance components revealed that fruit mass, CM, and wax mass and strain of the CM had a high genotypic variance and a low residual error variance. Stomatal density ranged from 0.12 stomata/mm2 in ‘Adriana’ to 2.13 stomata/mm2 in ‘Namosa’. The heritability of stomatal density was 67.5%. Across cultivars and years, mean densities of microcracks were of similar orders of magnitude as those of stomata, but ranges were larger and the heritabilities of microcrack density lower. Permeability for transpiration was lowest in ‘Flamingo Srim’ and highest in ‘Nadino’; that for osmotic water uptake was lowest in ‘Adriana’ and highest in ‘Hedelfinger’. Heritability estimates for permeabilities were low. Based on these data, breeding strategies for less cracking susceptible fruit should focus on genotypes that maintain an intact CM throughout development. This may be achieved by selecting for low CM strain and high CM thickness because thicker CM have more “reserve” for thinning. Finally, genotypes that deposit cutin and wax also during Stage III would be most interesting but were not found among the cultivars investigated.