Characterization of microcracks in the cuticle of developing sweet cherry fruit

By S. Peschel, M. Knoche. 2005

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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 fl uorescence microscopy following infi ltration with a fl uorescence 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 ʻSam ̓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 ʻHedelfi nger ̓(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 fi rst 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 fi rst 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. Rain-cracking of fruit is a limitation in production of sweet cherry fruit worldwide. Cracking is thought to result from increased turgor caused by water uptake into the fruit (Andersen and Richardson, 1982; Considine and Kriedemann, 1972). Macroscopic fruit cracking is preceded by development of minute fractures in the cuticular membrane (CM) that initially traverse only the CM and that do not extend into epiand hypodermal cell layers and the mesocarp (Glenn and Poovaiah, 1989). We refer to these initial fractures of the CM as microcracks, since they are not detected without the aid of a microscope (Glenn and Poovaiah, 1989). These microcracks are often observed on immature and mature sweet cherry fruit (Glenn and Poovaiah, 1989; Sekse, 1995) and frequency of microcracks generally increases during development (Knoche et al., 2001). Development of microcracks may be an early event in macroscopic fruit cracking, since 1) the CM is the rate limiting barrier to water transport across the fruit surface (Beyer and Knoche, 2002; Knoche et al., 2000) and water uptake was increased in fruit having microcracks (Glenn and Poovaiah, 1989), and 2) macroscopic cracks were reported to develop along microcracks and progress along their length (Glenn and Poovaiah, 1989). Received for publication 2 Mar. 2005. Accepted for publication 7 May 2005. This research was funded in part by grants from the Deutsche Forschungsgemeinschaft, the Saale Obst Erzeugerund Absatzgenossenschaft and the Gisela Foundation. We thank Kurt Ehm, Wolfgang Meyer, and Erhard Sonnenkalb for providing fruit samples; Matthias Hinz and Evelyn and Ruth Richter for technical assistance; and John Bukovac, Greg Lang, and Peter Petracek for critical comments on an earlier version of this manuscript. 1To whom reprint request should be addressed. E-mail address: moritz. knoche@landw.uni-halle.de Up to now the mechanistic basis of formation of microcracks in the CM is unknown. Knoche et al. (2004) demonstrated that the CM is markedly strained particularly during stage III or “fi nal swell” of fruit development. This stage is characterized by a rapid increase in fruit mass and hence surface area, while CM mass on a per fruit basis remains constant. The strain of the CM resulting from fruit surface expansion in the absence of CM deposition is expected to cause stress and stress induced failure is a basic concept in material science. To gain a better understanding of the mechanism of formation of microcracks in the CM, analysis of the fracture pattern may be helpful. If strain was an important factor, crack formation should be related to fruit surface expansion and development of strain in the CM. Furthermore, microcracks would be expected to be preferentially oriented relative to the primary axis of strain. Finally, formation of microcracks in the CM should result in an increase in strain of the underlying epidermal cells. To test this hypothesis, we investigated the occurrence, distribution and characteristics of microcracks in the CM of developing sweet cherry fruit. We focused on developing sweet cherry fruit, since cracking susceptibility increased markedly towards maturity (Christensen, 1996). Materials and Methods PLANT MATERIAL. Frequency and distribution of microcracks in the CM of developing sweet cherry fruit (ʻHedelfi ngerʼ, ̒ Kordiaʼ, ʻSamʼ, and ʻVanʼ; all grafted on P. avium ʻAlkavo ̓rootstocks planted in 1989) CM were monitored. Fruit were sampled at weekly intervals between 16 and 92 DAFB (ʻHedelfi ngerʼ), 16 and 85 DAFB (ʻKordiaʼ), 16 and 78 DAFB (ʻSamʼ), and 16 and Book 1.indb 487 6/13/05 3:25:59 PM 488 J. AMER. SOC. HORT. SCI. 130(4):487–495. 2005. 71 DAFB (ʻVanʼ) in a commercial orchard located near Eisleben, Germany (lat. 51°31 ́N, long. 11°44 ́E) in 2000. These fruit originated from a minimum of ten representative trees per cultivar and sampling time. Fruit were selected for uniformity and freedom of visual defects, pooled per cultivar, transferred to the laboratory and held overnight at 5 °C. Subsamples were formed and used in our earlier study for determining fruit mass, osmotic potential, strain of CM, and mass of CM per unit fruit surface area (Knoche et al., 2004) and in the present study for monitoring frequency and distribution of microcracks in the CM. Sweet cherry fruit of ʻBurlat ̓(grafted on P. avium ʻAlkavoʼ) were obtained from a commercial orchard at Höhnstedt, Germany (lat. 51°30 ́N, long. 11°44 ́E) in the 2003 growing season and used for characterizing microcracks. Trees were cultivated according to current regulations for integrated fruit production (Landespfl anzenschutzamt Sachsen-Anhalt, 2000, 2003). MASS AND SURFACE AREA OF DEVELOPING FRUIT. Data for mass of developing ʻHedelfi ngerʼ, ʻKordiaʼ, ʻSamʼ, and ʻVan ̓ fruit were reproduced and redrawn with permission from Knoche et al., Physiol. Plant. 120:667–677, 2004. Fruit surface area was calculated from fruit mass assuming a spherical shape of the fruit and a density of 1000 kg·m–3 as a fi rst approximation. MONITORING MICROCRACKS IN DEVELOPING FRUIT. Following equilibration at room temperature, fruit were incubated in a solution containing the fl uorescent tracer acridine orange at 0.1% and a silicone surfactant [surface tension, 21 mN·m–1 at 0.1% (Silwet L-77; Witco, Düsseldorf, Germany) (Knoche et al., 1991)] in 50 mM citric acid buffer at pH 6.5. Solutions containing Silwet L-77 infi ltrated microcracks in the CM. After 1 to 2 min, fruit were removed from solution and blotted dry using tissue paper. Exocarp segments (ES) comprising CM, epidermis, hypodermis and some cell layers of mesocarp tissue were excised by razor blade from cheek, suture, pedicel cavity and stylar regions (ʻSamʼ; 22 to 78 DAFB) and from cheek and stylar regions (ʻHedelfi ngerʼ, 22 to 92 DAFB; ̒ Kordiaʼ, 22 to 85 DAFB; ʻVanʼ, 22 to 71 DAFB). ES were transferred to the stage of a fl uorescence microscope (model BX-60; Olympus, Hamburg, Germany) and viewed at ×100 (330–385 nm excitation wave length, 420 nm emission wave length; Olympus fi lter module U-MWU). Ten randomly selected areas (621 × 468 μm2) per ES of a total of ten fruit per sampling date, region and cultivar were inspected for microcracks in the CM. Severity of microcracks was quantifi ed using the following rating scheme (Fig. 1): score 0, surface without cracks (Fig. 1A); score 1, single microcrack Fig. 1. Fluorescence micrographs of the sweet cherry fruit surface. (A) Intact fruit surface with stoma and (B–F) microcracks in the cuticle. Images (A–E) are representative for scores of rating scheme used to assess severity of cracking. (A) No microcrack (score 0). (B) Single microcrack shorter than longest dimension of underlying epidermal cell (score 1). (C) More than one microcrack shorter than longest dimension of underlying epidermal cell or single microcrack longer than longest dimension of epidermal cell (score 2). (D) Less than eight microcracks longer than longest dimension of underlying epidermal cells (score 3). (E) Eight or more microcracks longer than longest dimension of underlying epidermal cells (score 4). (F) Crack formation in relation to scratches on the fruit surface. Images were taken at ×100. Scale bar 100 μm. Book 1.indb 488 6/13/05 3:26:03 PM 489 J. AMER. SOC. HORT. SCI. 130(4):487–495. 2005. shorter than longest dimension of the underlying epidermal cell (Fig. 1B); score 2, more than one microcrack shorter than longest dimension of underlying epidermal cell or single microcrack longer than longest dimension of the epidermal cell (Fig. 1C); score 3, less than eight microcracks longer than longest dimension of underlying epiderm

Subfields: Molecular Biology, Plant Science

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