Fig 1.
Schematic illustrating the procedure used to quantify crack number and crack length on the six orthogonal regions of the fruit surface.
A) Top view of a fruit with receptacle and pedicel in the center of a circle. B) Front view of a fruit illustrating the pedicel cavity and stylar scar regions. The pedicel cavity region was approximated by a circle with 0.55 times the diameter of the fruit and a (half) torus having a radius defined as height h1. The stylar scar region was approximated by the cap of a sphere of height h2. C) Side view of a fruit with four trapezoids (only one is visible) representing the cheek, the suture and the two shoulders of a fruit. The trapezoids were drawn such that their heights were delineated by the base of the torus marking the pedicel cavity region, and their bases by the cap delineating the stylar scar region. The vertical margins of each trapezoid were delineated by the respective width of the fruit from which a 10% boundary was subtracted to minimize errors due to curvature of the surface. D-F) Representative images taken from a video clip at 0 h (D), 13 h (E) and 24 h (F) of the suture region of a ‘Regina’ sweet cherry when incubated in deionized water. Scale bar = 5 mm.
Fig 2.
Time course of macroscopic cracking in different regions of the fruit surface of ‘Kordia’ sweet cherry.
A,C,E. Numbers of macrocracks (A), average lengths of macrocracks (C), and cumulative length of macrocracks (E) in stylar scar and pedicel cavity regions. B,D,F. Number of macrocracks (B), average lengths of macrocracks (D) and cumulative lengths of macrocracks (F) in cheek, suture and shoulder regions. Fruit were incubated in deionized water to induce cracking.
Table 1.
Number of cracks and cumulative length of cracks per fruit in various sweet cherry cultivars.
Fig 3.
Long term (A) and short term time courses (B) of change in rate of extension of macrocracks (main graphs) and in length of individual macrocracks (insets) in ‘Kordia’ sweet cherry. Fruit were incubated in deionized water to induce cracking.
Fig 4.
Micrographs of macroscopic cracks (macrocracks) and microscopic cracks (microcracks) in the skin of ‘Regina’ sweet cherry.
A) Close up view of the tip of an extending macrocrack. B) Light micrograph of the tip of a macrocrack in the cuticle of the skin of a mature sweet cherry fruit depicting zones I, II and III of a developing macrocrack. C) Light micrograph of fruit skin in zone I. Zone I is the zone ahead of a developing crack with an intact cuticle and adherent, still-living, epidermal cells. D) Light micrograph of fruit skin in zone II. Zone II represents the tip of the microcrack (indicated by the white arrow) showing rupture of the cuticle. In zone II, the first separation of epidermal cells occurs, some of which are dead. E) Light micrograph of fruit skin in zone III. In zone III, the microcrack develops into a macrocack that extends deep into the epidermal and hypodermal cell layers and begins to gape. Essentially all the cells along a macrocrack are dead. Bars in A = 1 mm, B = 50 μm, C-E = 20 μm.
Fig 5.
Time course of cell wall swelling (A) and the proportions of living vs. dead cells (B) along a developing macrocrack. C) Relationship between cell wall thickness and percentage of living cells. ‘Regina’ fruit were incubated in deionized water and removed from solution at different times to investigate the development of a skin macrocrack. Zone I represents the intact, healthy skin ahead of a macrocrack at time zero (t = 0 h), zone II the tip of a macrocrack at t = 6 h and zone III a macrocrack where gaping has begun. The macrocrack is now bordered by dead cells.
Table 2.
Cell wall thickness in different zones of a developing macrocrack in the fruit skin.
Table 3.
Cell wall thickness in different zones of a macrocrack in the cheek, pedicel cavity and stylar scar regions of ‘Regina’ sweet cherry.
Table 4.
Effect of malic acid on cell wall thickness in different zones of a developing skin macrocrack of ‘Regina’ sweet cherry.
Fig 6.
Composite of micrographs of ‘Burlat’ sweet cherry fruit that macrocracked in the stylar scar region during incubation in deionized water.
Images were obtained following staining with calcofluor white or following binding of monoclonal antibodies (mAbs) against epitopes on cell walls exposed in the macrocrack surface. The mAbs reacting with epitopes of hemicelluloses were LM11 (anti-xylan/arabinoxylan), LM21(anti-mannan) and LM25 (anti-xyloglucan). The mAbs against pectin epitopes were LM5 (anti-galactan), LM6 (anti-arabinan), LM7 (anti-homogalacturonan), LM8 (anti-xylogalacturonan), LM19 (anti-homogalacturonan) and LM20 (anti-homogalacturonan). The first column of the composite was obtained under bright field illumination, the second column following calcofluor white staining (CFW) using UV light and the third column under fluorescent light following mAbs binding. All images 0.8x. Scale bar = 1 mm.
Fig 7.
Composite of micrographs of ‘Samba’ sweet cherry fruit that had macrocracked in the stylar scar region during incubation in deionized water.
Images were taken following staining with calcofluor white (CFW) or following binding of monoclonal antibodies (mAbs) against epitopes on cell wall surfaces exposed in macrocracks. The mAbs reacting with epitopes of hemicelluloses were LM6 (anti-xylan/arabinoxylan), LM8 (anti-xylogalacturonan), LM19 (anti-homogalacturonan), and LM20 (anti-homogalacturonan). Images were viewed under bright field illumination (BF), under UV (CFW) or under fluorescent light (mAb). Scale bars in the first, second and third columns = 1 mm, in the fourth and fifth columns = 0.2 mm.
Fig 8.
Composite of micrographs of ‘Burlat’ sweet cherry fruit that cracked in the stylar scar region during incubation in deionized water.
A) Bright field image (BF). B) Same specimen as A, but macrocracks stained using calcofluor white (CFW) and viewed under UV light. Same specimen as A, but now treated with the monoclonal antibodies LM19 (anti-homogalacturonan) and viewed under fluorescent light. D) Detailed view of cracks labeled with LM19 showing crack network with a microcrack (‘zone II’) and a developing macrocrack (‘zone III’). Scale bars = 1 mm (A-C) or 100 μm (D). In the microcrack (zone II), the cuticle has fractured, but epidermal cells are largely intact. The mAb LM19 labeled the periclinal cell walls. In the macrocrack in zone III separation of epidermal cells along their anticlinal cell walls began near the tip. Separation proceeded along the macrocrack and gaping began (indicating release of stress and strain).
Fig 9.
Composite of micrographs of ‘Burlat’ sweet cherry fruit that had cracked in the stylar scar region during incubation in deionized water.
Images were taken following staining with calcofluor white (CFW) or following binding of the monoclonal antibody (mAb) 2F4 in the absence (2F4, -CaCl2) or presence of Ca (2F4, +CaCl2). The mAb 2F4 identifies dimeric associations of homogalacturonan chains with Ca2+. Images were viewed under bright field (BF), in UV (CFW) or in fluorescent light (mAb). All images at 0.8x. Scale bars = 1 mm.
Fig 10.
Sketch illustrating the sequence of events in macroscopic rain-cracking of sweet cherry.
The increase in surface area in the absence of cuticle deposition [5] causes tension [7] and formation of microcracks [6]. Microcracking is aggravated by surface moisture [50]. Microcracks impair the cuticle’s barrier function [50] and focus water uptake in a particular region of the fruit surface [30]. Water penetration causes individual cells to burst, probably in the outer mesocarp where the osmotic potential is more negative than in the skin [41]. Malic acid leaks into the apoplast causing further leakage of membranes in surrounding cells and the extraction of Ca2+ from the cell wall [29]. Cells plasmolyse and collapse. Due to the loss of turgor, cell walls swell [22]. Swelling results in a weakening of the pectin middle lamella, the loss of cell to cell adhesion and decreased fracture pressure [22]. The cells separate along the middle lamella (this paper), the crack propagates as the skin “unzips” like a ladder in knitted fabric. This model is referred to as the zipper model [30].