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.
Moritz Knoche and Stefanie Peschel
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.
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.
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.
Eckhard Grimm, Stefanie Peschel, and Moritz Knoche
Mottling (pale spots) is clearly visible to the naked eye in all regions of the surface in all except for yellow cultivars of sweet cherry fruit (Prunus avium L.). The objective was to characterize these spots and their distribution on the exocarp. Within the spots, anthocyanins were limited to the epidermal cell layer but, in areas immediately adjacent to the spots, anthocyanins were present in the epidermal and in the hypodermal cell layers (making these areas darker). In ‘Sam’ sweet cherries, the median length and width of a spot in the cheek region were 390 and 162 μm, respectively, and the median area was 0.053 mm2 per spot. The spatial density in the cheek region averaged 1.94 (± 0.13) spots per mm2 and the percentage of surface area covered by the spots was 12.5% (± 1.07%). Epidermal cells within a spot had slightly larger projected surface areas than those in the adjacent region and thicker cell walls. The margins of the spots did not align with the anticlinal walls of the epidermal cells. The spots’ long axes were oriented parallel with the stem/stylar scar axis, whereas the slightly elongated epidermal cells within and adjacent to the spots were orientated perpendicular to the stem/stylar scar axis. The spatial density of spots and the cumulative spot area were highest in the region of the stylar scar, intermediate in the cheek and stem cavity, and lowest in the suture region. Spot spatial density on small fruit exceeded that on larger fruit, but the areas of individual spots was smaller. When an exocarp segment was excised from the cheek of a fruit, it contracted slightly as elastic strain was released. The projected surface area of the spots and that of the whole segment decreased to a similar extent. Our data suggest that spots result from a tensional failure during Stage III development in which the anthocyanin-containing hypodermal cell layer tears (schizogenously) and separates from the epidermis. This being the case, the pale spots (mottling) can be referred to as “strain spots.”
Marco Beyer, Stefanie Peschel, Moritz Knoche, and Manfred Knörgen
Water uptake in different regions of the sweet cherry fruit (Prunus avium L. cv. Sam) was investigated following selective application of silicone sealant to the pedicel end, pedicel cavity, pedicel/fruit juncture, or stylar scar of detached fruit. The time course of water uptake was monitored gravimetrically during a 3-hour incubation period in deionized water (20 °C). Sealing the pedicel end and/or pedicel/fruit juncture significantly reduced rates and total amount (3 hours) of water uptake, but sealing the stylar scar had no effect. The amount of water penetrating via the pedicel/fruit juncture increased between 50 and 85 days after full bloom. During the same period the maximum force required to detach pedicels from fruit (fruit removal force) fell from 5.2 ± 0.5 to 2.1 ± 0.2 N. The amount of water penetrating via the pedicel/fruit juncture and the fruit removal force were negatively related. Nuclear magnetic resonance (NMR) imaging of mature fruit incubated in D2O indicated that D2O accumulated in the pedicel cavity region and the pedicel. Our data suggest that the pedicel end and pedicel/fruit juncture, but not the stylar scar, are regions of preferential water uptake in detached fruit. Chemical name used: deuterium oxide (D2O).
Eckhard Grimm, Stefanie Peschel, Tobias Becker, and Moritz Knoche
Rain-cracking of sweet cherry (Prunus avium L.) fruit involves failure of the exocarp caused by excessive stress and strain. The objective of our study was to quantify exocarp strain in developing cherries. The release of linear elastic strain was followed in vivo using a gaping assay, whereas the release of biaxial elastic strain was followed in vitro after excision of small exocarp segments (ESs) that were submerged in silicone oil and strain release quantified by image analysis. When mature sweet cherry fruit were cut (by making two or more deep, longitudinal incisions parallel to the stylar/pedicel axis and on opposing sides of the fruit down to the pit), the incisions rapidly “gaped.” The gaping wounds continued to widen as they progressively released the linear elastic strain in the skin. By 24 hours the combined widths of two gapes represented 8.8% ± 0.1% of the fruit circumference. Increasing the number of cuts from two to 12 increased the cumulative gape widths to 14.9% ± 0.2%. In ES, monitoring the time course of relaxation after excision revealed a rapid release of biaxial strain, having a half-time of ≈2.7 minutes. Relaxation continued, but at a decreasing rate, for up to 48 hours. Across eight cherry cultivars, the biaxial strain in the exocarp at maturity ranged from 18.7% ± 1.9% in ‘Lapins’ to 36.0% ± 1.8% in ‘Katalin’. Elastic strain in the ES was always lower than that measured in an isolated cuticular membrane (CM). Increasing the temperature from 2 to 35 °C increased the rate of strain release and also the total percent strain released at 96 hours. In developing ‘Hedelfinger’ sweet cherry fruit, there was essentially no elastic strain in the exocarp at 45 days after full bloom (DAFB). Thereafter, significant elastic strain developed, reaching a maximum of 47.6% ± 2.5% at 87 DAFB. The effect of exocarp cell turgor on strain in the ES (evidenced by the difference in the reversible strain between ES with and without turgor) was closely and positively related to the relative area growth rate of the skin (r 2 = 0.957). Strain release peaked at ≈59 DAFB, and there was no effect of turgor on strain release in mature fruit. Our data demonstrated the following: 1) the exocarp is a viscoelastic material composite; 2) at maturity, plastic and elastic strain components make up 66% and 34% of the total percent strain, respectively; 3) elastic strain in the exocarp increases during Stage III development; and 4) the strain in the exocarp is unaffected by strain in the CM. Thus, the epidermis and hypodermis layers must represent the main, load-bearing structure in sweet cherry fruit with the cuticle making a mechanically insignificant contribution.
Andreas Winkler, Stefanie Peschel, Kathleen Kohrs, and Moritz Knoche
Rain cracking of sweet cherry (Prunus avium L.) fruit is commonly thought to result from excessive net water uptake. This excess increases flesh turgor, which then strains and eventually ruptures the skin at the weakest point. This idea—the critical turgor hypothesis—assumes the fruit comprises a semifluid flesh, held under pressure by a taut skin. The objectives of this study were to test the validity of this popular hypothesis. We investigated the effects of 1) the different pathways of water uptake and 2) the fruit’s water balance on cracking. Incubating fruit of 19 cultivars in water resulted in rapid fruit cracking. The time to 50% cracking (T50) averaged 7.5 ± 1.3 hours with considerable variability between cultivars (T50 range from 1.5 to 18.6 hours). The amount of water taken up at 50% cracking (WU50) averaged 96.5 ± 17.6 mg (WU50 range from 17.7 to 331.5 mg). There was no correlation between either the T50 or the WU50, and the rate of water uptake. Also, there was no correlation between the values of T50 (r = 0.58) and only a weak correlation between the values of WU50 (r = 0.80*) determined in different years. Comparing the value of WU50 under incubation vs. under perfusion revealed a 3.9- to 38-fold higher WU50 under perfusion (397.6 to 1840 mg) than under incubation (48.8 to 102.6 mg). This marked dissimilarity remained, regardless of pretreatments with isotonic polyethylene glycol (PEG) 6000 to induce microcracking or by manipulation of skin wetness during perfusion. Sealing the pedicel/fruit junction markedly decreased the rate of water uptake under incubation. It had no effect on the T50, and it markedly decreased the WU50. Similarly, manually induced skin defects greatly increased the rate of water uptake but, with few exceptions, had no effect on the T50, whereas, the WU50 had increased. The location on the fruit surface of the resulting cracks was not related to the region of the skin in which the manual defect was induced. Allowing the fruit to transpire increased both, the T50 and the WU50. Interestingly, the amount of water lost by transpiration exceeded the amount that was subsequently required to cause cracking up to 5-fold. Incubating fruit with their stylar ends immersed in water, whereas their remaining surfaces were in air of 0%, 28%, 75%, or 100% relative humidity (RH) resulted in net losses of water of up to 5.9 ± 0.7 mg·h−1, nevertheless their stylar ends still cracked. All our results indicate rain cracking in sweet cherries is a localized phenomenon that is not related to the net fruit water balance (the critical turgor hypothesis) but is the result of more local exposure of the fruit skin to liquid-phase water (the zipper hypothesis).
Holger Weichert, Stefanie Peschel, Moritz Knoche, and Dieter Neumann
Recent studies established that some ferric salts, including FeCl3, decrease water permeability of the sweet cherry (Prunus avium L.) fruit exocarp and fruit cracking, presumably by a pH-dependent precipitation reaction that blocks high-flux pathways across the fruit surface. The objectives of our study were the following: to establish the effect of receiver pH on penetration of 55FeCl3 through excised exocarp segments (ES) and isolated cuticular membranes (CM) and to localize any Fe precipitates in the epidermal system of mature sweet cherry fruit. Penetration was studied using an infinite dose diffusion system where 55Fe penetrated from donor solutions of ferric salts (10 mm, pH 2.2–2.6) or EDTA-Na-Fe(III) (10 mm, pH 5.0) across an interfacing ES or CM into aqueous receiver solutions of pH values ranging from 2.0 to 6.0. For receiver pH 2.0, 55Fe penetration of the ES from a 10 mm FeCl3 donor (pH 2.6) was linear with time, but for receiver pH ≥ 3.0, penetration was low and insignificant. Increasing the pH of the water receiver from 2.0 to 6.0 in the course of an experiment resulted in an immediate halt of penetration regardless of whether 55Fe penetration occurred from FeCl3 (pH 2.6), Fe(NO3)3 (pH 2.6), or Fe2(SO4)3 (pH 2.4) as donor solutions (all at 10 mm). Only from EDTA-Na-Fe(III) (pH 5.0) 55Fe penetration continued to occur albeit at a decreased rate (−30%). At receiver pH 2.0, the 55FeCl3 flux through stomatous ‘Sam’ ES averaged 10.4 ± 2.3 pmol·m−2·s−1 and was positively correlated to stomatal density. Conventional and analytical electron microscopy (energy dispersive X-ray analysis, electron spectroscopic imaging, and electron energy loss spectroscopy) identified ferric precipitates in periclinal and anticlinal cell walls of epidermal cells underlying the cuticle, but not within the cuticle. These data indicate that the lack of 55Fe penetration from donor solutions of ferric salts through the ES into a receiver solution at pH ≥ 3 and the previously reported decrease in water uptake and cracking as a response to immersing fruit in solutions of ferric salts are the result of a precipitation reaction at the cuticle/cell wall interface in the sweet cherry exocarp. Although spray application of ferric salts is prohibitive for ecotoxicological reasons, understanding their mechanism in decreasing water uptake and fruit cracking may be helpful in the search for alternate compounds that are effective and ecotoxicologically acceptable.
Holger Weichert, Carina von Jagemann, Stefanie Peschel, Moritz Knoche, Dieter Neumann, and Wilfried Erfurth
Water uptake through the exocarp of intact sweet cherry [Prunus avium (L.)] fruit was determined gravimetrically in an immersion assay (25 °C). Fruit with sealed pedicel/fruit juncture were incubated in water during the first interval (0 to 0.75 hour) and in 10 mm salt solutions of selected cations during the second (0.75 to 1.5 hours) and third interval (1.5 to 2.25 hours) of an experiment. Rates of water uptake (F) were calculated for first, second and third intervals (FI, FII and FIII, respectively) and salt effects indexed by the ratios FII/FI and FIII/FI. AgNO3 (FII/FI = 0.65), NaCl (0.70), BaCl2 (0.67), CdCl2 (0.69), CuCl2 (0.42), HgCl2 (0.58), and SrCl2 (0.69), and the salts of trivalent cations AlCl3 (0.50), EuCl3 (0.58), and FeCl3 (0.49), significantly decreased water uptake into mature `Sam' fruit as compared to the water control (0.87). KCl (0.82), NH4Cl (0.85), CaCl2 (0.75), MgCl2 (0.88), MnCl2 (0.81), and ZnCl2 (0.72) had no effect, LiCl (1.00) increased uptake. Similar data were obtained for FIII/FI. The effect of FeCl3 on water uptake was independent of the presence of CaCl2, AlCl3, or CuCl2, as sequential or simultaneous treatment with these salts reduced water uptake to the same extent as with FeCl3 alone. Increasing FeCl3 concentration up to 1 mm decreased uptake, higher concentrations had no further effect. FeCl3 and CaCl2 to a smaller extent decreased water uptake in developing `Regina' sweet cherry fruit (55 to 91 days after full bloom). FeCl3 had no significant effect on water uptake along the pedicel/fruit juncture, but markedly reduced uptake through the exocarp of all cultivars investigated (`Burlat', `Early Rivers', `Hedelfinger', `Knauffs', `Regina', `Sam', `Summit', and `Van'). Effects of CaCl2 on water uptake were limited to `Burlat', `Early Rivers', and `Hedelfinger'. CaCl2 and FeCl3 both decreased fruit cracking, but FeCl3 was more effective. The mode of action of mineral salts in decreasing water uptake and fruit cracking and their potential for field use are discussed.