Increase in Epidermal Planar Cell Density Accompanies Decreased Russeting of ‘Golden Delicious’ Apples Treated with Gibberellin A4+7

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Eric CurryTree Fruit Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 1104 N. Western Avenue, Wenatchee, WA 98801

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A 2-year study was conducted in a ‘Golden Delicious’ (Malus ×domestica Borkh.) orchard with a high incidence of physiological fruit russeting to examine the effect of gibberellin A4+7 (GA4+7) on apple epidermal cell size. Beginning at petal fall, four sequential applications of GA4+7 (0, 15, or 30 mg·L−1) were applied to whole trees every 7–10 days with an orchard air-blast sprayer at a volume of ≈1000 L·ha−1. Fruit epidermal tissue samples were taken approximately monthly beginning 1 week after the fourth application. Tissue was treated in the laboratory with an enzyme mixture to remove cellular debris in preparation for examination using either light or scanning electron microscopy (SEM). In 2007, because russeting was insignificant, treatment differences could not be established. Moreover, transillumination microscopy did not permit accurate measurement of ‘Golden Delicious’ fruit epidermal planar cell area beyond midseason because the isolated cuticle became thick and multilayered. In 2008, however, respective treatments of 15 and 30 mg·L−1 reduced calyx end russeting by 40% and 83% and increased epidermal planar cell density by 14% and 27% as measured using SEM.

Abstract

A 2-year study was conducted in a ‘Golden Delicious’ (Malus ×domestica Borkh.) orchard with a high incidence of physiological fruit russeting to examine the effect of gibberellin A4+7 (GA4+7) on apple epidermal cell size. Beginning at petal fall, four sequential applications of GA4+7 (0, 15, or 30 mg·L−1) were applied to whole trees every 7–10 days with an orchard air-blast sprayer at a volume of ≈1000 L·ha−1. Fruit epidermal tissue samples were taken approximately monthly beginning 1 week after the fourth application. Tissue was treated in the laboratory with an enzyme mixture to remove cellular debris in preparation for examination using either light or scanning electron microscopy (SEM). In 2007, because russeting was insignificant, treatment differences could not be established. Moreover, transillumination microscopy did not permit accurate measurement of ‘Golden Delicious’ fruit epidermal planar cell area beyond midseason because the isolated cuticle became thick and multilayered. In 2008, however, respective treatments of 15 and 30 mg·L−1 reduced calyx end russeting by 40% and 83% and increased epidermal planar cell density by 14% and 27% as measured using SEM.

The presence of russet on ‘Golden Delicious' (Malus ×domestica Borkh.) apples has long been a concern to producers and marketers of fresh fruit throughout the world because it detracts from the smooth, uniform finish of the fruit and results in economic loss resulting from grade reduction (Faust and Shear, 1972a). Physical russetting is the result of irreversible injury to the peel by impact, abrasion, or repeated agitation by another material such as might occur with hail or limb and leaf rubbing. Physiological russeting, on the other hand, although foundationally genetic (Eccher et al., 2008), may be induced or aggravated by various factors including: 1) xenobiotics (Creasy and Swartz, 1981; Hatch, 1975; Lallu et al., 2010; McCormick and Streif, 2008; Sanchez et al., 1992); 2) changes in ambient temperature, humidity, and aridity (Creasy, 1980; Faust and Shear, 1972b; Fogelman et al., 2009; Meyer, 1944; Tukey, 1959); and 3) insects and pathogens such as rust mite, ring virus, and various bacteria, yeasts, and fungi (Daines et al., 1984; Duso et al., 2010; Easterbrook and Fuller, 1986; Heidenreich et al., 1997; Lindow et al., 1998; Wood, 2010). In the orchard, these factors often are related and/or codependent.

Many methods have been evaluated to alleviate physiological russeting in apple. Like other environmentally based peel disorders such as superficial scald, sunburn, scarfskin, or flecking, a consistent response to treatment is often difficult to achieve or predict. One of the more successful methods of reducing physiological russeting is use of multiple topical applications of gibberellins during early fruit development. Since the first published reports over 35 years ago (Eccher, 1975; Eccher and Boffelli, 1978; Edgerton and Veinbrants, 1979; Taylor, 1975), researchers have conducted numerous studies to better understand the mechanisms underlying the beneficial effects of gibberellins on apple peel finish.

Eccher (1975) first observed less irregularity among epidermal cells and reduced cuticle cracking in fruit treated with GA4+7 than in untreated fruit. Skene and Greene (1982) recognized the relationship between microcracking in the cuticle and regions of russeting in ‘Cox's Orange Pippin’. [Also, see the review regarding fruit skin splitting and cracking (Opara et al., 1997).] Microcracking in ‘Golden Delicious’ peel tissue was reported to increase as a result of surface wetting (Knoche and Grimm, 2008). More recently, Knoche et al. (2011) found water-induced russeting and microcracking of ‘Golden Delicious’ apples decreased on fruit pretreated with multiple applications of GA4+7. They also reasoned the effect of GA4+7 on microcracking and, therefore, russeting must reside with the epidermal and hypodermal cell layers.

Bukovac and Nakagawa (1968) first reported increases in size and number of cortex cells in apples treated with high rates of GA4+7 in lanolin paste applied 2 weeks after anthesis. To my knowledge, the effect on apple epidermal cells, of multiple applications of GA4+7, has not been quantitatively examined. Thus, the objective of this work was to determine whether the decrease in russetting of ‘Golden Delicious’ apples from multiple applications of GA4+7 was accompanied by an increase in epidermal planar cell density (number of epidermal cells per unit peel surface area).

Materials and Methods

Experiments were conducted in 2007 and 2008 in a uniform, mature, commercial orchard of ‘Golden Delicious’/‘M.M.106’ (‘MM.106’) trees growing in loamy sand near Mattawa, WA (lat. 46°39′16.58″ N, long. 119°52′32.12″ W; elevation 192 m). Trees were irrigated by undertree impact sprinklers and fruit from trees in most areas within this 13.2-ha block had a 70% to 80% historical incidence of russeting (≈7 of 10 years 50% or greater of the harvested fruit had calyx and/or shoulder russeting sufficient to cause reduction in grade).

Treatment dosages of 0, 15, or 30 mg·L−1 GA4+7 (ProVide®; Valent Biosciences Corp., Libertyville, IL) were applied topically to whole trees at first petal fall (FPF) and every 7–10 d thereafter for four sequential applications. Each treatment was applied to three rows of ≈20 trees per row with an orchard air-blast sprayer at a volume of ≈1000 L·ha−1. Treatment at the rate of 0 mg·L−1 was water only. Two untreated buffer rows separated each treatment row in a randomized block design.

Fruit tissue sampling.

Fruitlet collection for peel tissue sampling began 1 week after the fourth (final) GA4+7 application. In 2007, the sampling dates were 8 June, 18 June, 17 July, and 17 Aug. In 2008, the sampling dates were 12 June, 16 July, 13 Aug., and 10 Sept. Four fruit of similar size on the east side of each of four trees within each treatment row were removed and handled only by the stem and calyx end so as not to disturb the epidermal tissue. During the first two sampling dates, whole fruit were too small to be held securely in foam-pocketed trays; thus, fiber trays were modified as follows. To hold the apple securely on the tray and keep it from rolling around and disturbing the surface, a small hole ≈5 mm in diameter was punched through the bottom of each cup on the fiber tray. The stem of the apple was pushed through the hole and clamped on the opposite side with a small binder clip to secure it snugly against the fiber tray. In July and August, fruit were large enough to fit snugly in foam cells with the interior side of the fruit (side facing the vertical axis of the tree trunk) facing up. Fruit fitted in foam cells were placed in fiber cartons with lids and transported to the laboratory.

Fruit sampling for russet evaluations.

At harvest, ≈3–5 d after the last tissue sample date, 10 apples of approximately the same size were removed from 10 trees from each treated row. Fruit were transported to the laboratory and kept at 1.1 °C until further examination. Each apple was rated for russet severity (percent of fruit surface) by location (stem end or calyx end) within 10 d of harvest.

2007: sample preparation for light microscopy.

In the laboratory, the diameter of each apple was measured across the widest part of the shoulder. Two fruit per tree closest to the mean fruit diameter within each treatment replication were selected for epidermal tissue excision and processing (n = 24). From each apple, a longitudinal slice 5–8 mm wide was cut from the middle of each whole fruit roughly perpendicular to the axis of greatest sun exposure. A single epidermal section 8 mm long and 2 mm thick was excised from opposite sides of each slice at the widest diameter. Both sections were placed into a single plastic vial containing 5 mL of an enzyme solution containing pectinases [Enzyme Commission (EC) 3.2.1.15], cellulases (EC 3.2.1.4), and pectin lyases (EC 4.2.2.10) (Sigma-Aldrich, St. Louis, MO) formulated according to the method of Ju and Bramlage (1999) to remove cellular material. This enzyme solution was kept at 23 °C and changed weekly for 3 weeks. After enzymatic removal of extracuticular debris, isolated cuticles were kept at 23 °C in distilled water containing 0.01% sodium azide (NaN3) as an aid in controlling microbial growth (Lichstein and Soule, 1943).

To prepare for imaging, isolated cuticles were rinsed for several minutes in clean distilled water. Each cuticle was then mounted in water on a glass slide with a glass coverslip and examined using a stereomicroscope (Model SZX12; Olympus Corp., Tokyo, Japan) fitted with a CCD digital camera (Model CoolSNAP cf; Photometrics, Tuscon, AZ). The ocular reticle (100 divisions) was calibrated with a 10-mm stage micrometer (pitch 0.1 mm) at the magnification used for image capture. After selecting a representative area, a brightfield image was recorded for further image processing. All cuticle sections were imaged using the same magnification.

Images were processed using Image-Pro Plus (Version 4.5; Media Cybernetics, Inc., Bethesda, MD). The 24-bit TIFF color images were separated into individual eight-bit RGB (red, green, blue) channels and the green channel selected for measurements. Gray-scale images were printed and quadrants drawn on each print. To minimize observer bias, the top right quadrant of each printed image was always used for counting individual cells. Two observers counted the number of individual cells within the chosen quadrant by placing a red dot on each counted whole cell and a green dot on each partial cell using a felt tip marker.

2008: sample preparation for scanning electron microscopy.

Additional tissue samples were taken every 7–10 d, beginning at petal fall, using fruit from untreated trees. After the last treatment application, samples were taken every 3–4 weeks on the dates indicated previously. Samples were processed similarly to those of the previous year with several modifications. For fruit 15 mm or less in diameter, the fruit was cut equatorially into three sections of equal (visually) thickness. The middle third of the fruitlet was quartered longitudinally. Individual quarter sections were then processed as described subsequently. From fruit greater than 15 mm in diameter, a longitudinal slice 5–8 mm wide was cut from the center of each whole fruit, roughly perpendicular to the axis of greatest sun exposure. A single epidermal section 8 mm long and 2 mm thick was excised from each (opposite) side of each slice at the widest diameter. One section was set aside momentarily for further processing in preparation for examination using SEM, and the other section was placed into a single plastic vial containing 5 mL of the enzyme mixture as described previously. Vials containing apple tissue sections in enzyme solution were shaken gently using an environmental shaker kept at 38 °C. After each week in enzyme solution, samples in vials were sonicated for 10 min in a water bath kept at 38 °C before changing the solution. This was repeated until the cuticle sections were clear or there was no discernible particulate matter in the solution after sonication. Isolated cuticles were rinsed for several minutes in clean distilled water before air drying on a clean glass slide with a glass coverslip to prevent excessive curling.

Evaluation of fruit cuticles using scanning electron microscopy.

Surface wax morphology was examined from the remaining section of peel tissue excised previously according to the method of Curry (2005) with the following modifications. From the center of each piece of peel tissue, a section of cuticle ≈4–5 mm in diameter and 0.2 mm thick was shaved by hand using a 0.012-mm thick double-edge stainless steel razor previously rinsed with acetone and air-dried to remove any residual oil. The shaved cuticle sections were fixed to a 24-mm aluminum stub using double-sided carbon tape by pressing the edges of the section onto the tape using a pair of fine-tipped tweezers under a stereomicroscope. The stub was placed in a small glass vacuum desiccator containing packaged silica gel and kept at 20 °C and 1.3 × 104 Pa for 48 h or until further treatment. The time between first fruit incision and placing tissue under low vacuum in the glass desiccator was less than 2 min.

Before SEM evaluation, mounted tissue was coated with a gold/palladium alloy using a Desk II cold sputter coater (Denton Vacuum Inc., Morristown, NJ) fitted with a tilting omni-rotating head. With the sample 47 mm from the gold/palladium target, a coating thickness of ≈20 nm was achieved after 70 s at 40 mA and 2.6 Pa. Coated samples were kept in a vacuum desiccator and held under low vacuum at 1.3 × 104 Pa and 20 °C until microscopically examined using a Tescan Vega-II Model 5136LM Scanning Electron Microscope (Tescan, s.r.o., Brno, Czech Republic) equipped with both secondary and back-scattered electron detectors. Unless otherwise noted, images were obtained at 10 kV and 7.4 × 10−3 Pa.

Similarly, isolated, air-dried cuticles from the enzymatic treatment were fixed onto the aluminum stub with the cortical side facing up. Tissue was then processed for SEM examination as previously described.

Epidermal cells were counted by printing the gray-scale SEM image and tagging individual cells with colored markers as previously described. Individual cell measurements were made using the SEM software (Vega) by outlining the cell “pocket” just inside the raised portion (cell imprint) of the cuticle with the drawing tool. The program calculated the perimeter and planar area of each epidermal cell imprint. At least 20 cells per image were measured from three areas of each isolated cuticle and the means and errors recorded. Analysis of variance was performed and graphs generated using Systat statistical software (Version 12.0; Systat Software, Inc., Chicago, IL). Error bars within each graph represent + or ± sem (se).

Results and Discussion

Fruit russeting.

In 2007, fruit russeting in the section of orchard used for this study was insufficient to establish treatment differences (data not shown). Because russeting across the region was less, this effect was likely related to climate. Nevertheless, epidermal cell counts were recorded so that planar cell density could be calculated and compared. In 2008, treatment differences in russet reduction were apparent. Most of the russeting occurred on the distal half of the fruit (calyx end) with 77%, 31%, and 13% of the fruit having more than 20% russetted peel for GA4+7 treatments concentrations of 0, 15, and 30 mg·L−1, respectively. At the highest dosage concentration of applied GA4+7, 74% of apples had no russeting.

Epidermal cell density by transillumination microscopy, 2007.

A gray-scale image of the green channel (RGB) from a representative brightfield image similar to those from which planar epidermal cell density was determined for the three treatments in 2007 is shown in Figure 1. After examining all three channels, the green channel was chosen because of its slightly improved clarity (data not shown). The white dotted lines indicate the quadrant from which cells were counted. Obviously, the lines transected many cells. Therefore, transected cells were counted as half and the number of half cells was divided by two and added to the number of whole cells in the quadrant. Subsequent analysis indicated the planar cell density (number of cells per mm2) determined by this “half-cell” method was within 14% of the planar cell density determined by counting only the number of whole cells within the same approximate quadrant and then determining the exact area encompassing the cells to use as the divisor for density calculations (data not shown).

Fig. 1.
Fig. 1.

Gray-scale image of the green channel (RGB) from a representative brightfield TIFF image similar to those from which planar epidermal cell density was determined for gibberellin A4+7 treatments on ‘Golden Delicious’ apple in 2007. Dotted lines indicate quadrant from which number of cells was counted. Bar is 10 μM.

Citation: HortScience horts 47, 2; 10.21273/HORTSCI.47.2.232

Planar cell densities for ‘Golden Delicious’ epidermal tissue 1 week after the fourth GA4+7 treatment are shown in Figure 2. Analysis of variance indicated no difference between observers. Only results from Observer B indicated a difference among treatments; namely, the highest treatment rate resulted in the greatest epidermal planar cell density. It is likely there was insufficient definition in the images from which to gather accurate data verifying treatment differences, although the pattern of increasing cell density with increasing treatment rate was suggested by data from both observers. Indeed, this was the only sample date in 2007 from which data were recorded. Attempts to count cell number using cuticles isolated on subsequent sample dates failed because the individual cell definition using this method was poor (data not shown). This may have occurred as a result of 1) thickening cuticle + external amorphous wax layers attenuating and scattering the transmitted light; 2) ineffective or insufficient removal of cellular material; and/or 3) inability to identify newly forming cell imprints because of the thinness of the cutin outlining the new cells. That is, when epidermal cells divide, presumably each cell generates its own cutin pocket, and with this method of imaging, thickness of this pocket was insufficient to reduce transmitted light and generate the contrast necessary to identify newly forming cells. Nevertheless, the pattern of increasing number of smaller cells with increasing dosage of GA4+7 was sufficient to warrant a second attempt with improved cuticle isolation methodology and enhanced imaging, thereby affording greater magnification and detail resolution. (The transillumination method together with improved enzymatic removal of cellular material would be a useful alternative in this type of work where a SEM is not available.)

Fig. 2.
Fig. 2.

Graph of planar cell density vs. treatment application rate (two independent observers) from ‘Golden Delicious’ apple epidermal tissue sampled on 8 June 2007 and enzymatically treated to remove cell debris. Bars indicate + se.

Citation: HortScience horts 47, 2; 10.21273/HORTSCI.47.2.232

Examination of cutin imprints by scanning electron microscopy, 2008.

Cuticles isolated and cleared of cellular material using the improved enzymatic treatment protocol proved to be superior with regard to identifying individual epidermal cell imprints using SEM (data not shown). Figure 3 shows such a representative cuticle section from untreated fruit together with the overlay of individual cell area measurement outlines. Using this technique, a graph of planar cell area and cell density was constructed for cuticles sampled on 18 June 2008, 1 week after the fourth application of GA4+7 (Fig. 4). Differences among treatments are clear. Epidermal planar cell density increased by 14% and 27%, whereas epidermal planar cell area decreased by 18% and 31% for application rates of 15 mg·L−1 and 30 mg·L−1, respectively.

Fig. 3.
Fig. 3.

Scanning electron micrograph of the interior surface (flesh side) of a representative section of untreated ‘Golden Delicious’ apple fruit cuticle sampled on 12 June 2008 and enzymatically treated to remove cell debris. Example of individual cell area measurement outlines are in black. Arrow indicates developing cutin from recent cell division. Bar is 10 μM.

Citation: HortScience horts 47, 2; 10.21273/HORTSCI.47.2.232

Fig. 4.
Fig. 4.

Graph of planar cell area and planar cell density vs. treatment application rate from ‘Golden Delicious’ apple epidermal tissue sampled on 12 June 2008 and enzymatically treated to remove cell debris. Scanning electron micrographs were used for measurements. Bars indicate + se.

Citation: HortScience horts 47, 2; 10.21273/HORTSCI.47.2.232

Not unexpectedly, there was greater variability in both planar cell area and planar cell density for the GA4+7 application rate of 15 mg·L−1 than for both higher and lower rates. Possibly, this is related to activation threshold. As the apple enlarges, 1) cuticle thickens and may reduce penetration of aqueous a.i.; and 2) number of epidermal cells beneath each microdroplet decreases, thereby reducing number of affected cells. This is made clearer by the data in Figure 5, which shows cutin imprints of epidermal cells during the first 30 d of fruitlet development. The area within the 100-μm diameter circle (e.g., microdroplet) encompasses fewer, albeit larger, epidermal cells as fruitlet diameter increases. Thus, less a.i. may contact fewer cells. Whether a threshold exists, however, or whether a threshold changes with development is not known. Other factors that may influence efficacy during the first few weeks of fruit development are ambient conditions that alter drying time, the presence of other topically applied compounds, and increased leaf area (reduced spray penetration). In this particular orchard, canopy development at the FPF stage was quite different from 3 weeks later (E. Curry, personal observation).

Fig. 5.
Fig. 5.

Graph of planar cell surface area vs. fruitlet diameter of untreated ‘Golden Delicious’ apple cuticles isolated during the first ≈5 weeks of fruitlet development. Inset scanning electron micrographs indicate representative cutin imprints of epidermal cells from which measurements were made. Bars indicate ± se.

Citation: HortScience horts 47, 2; 10.21273/HORTSCI.47.2.232

Inconsistency of response to plant growth regulators, and particularly to GA4+7, can be frustrating for growers and field staff. Previous work has shown the first or first two applications of GA4+7 are often quite effective for reducing russet (Elfving and Allen, 1987), whereas applying more than four applications has no benefit (Meador and Taylor, 1987; Steenkamp and Westraad, 1988). Consider, for example, if instructions are to apply material beginning at FPF and every 7–10 d thereafter, the time to apply four treatments ranges from FPF + 21 d to FPF +30 d. Indeed, given this flexibility, by FPF + 14 d, one might have applied one or two additional applications and by FPF + 21 d, two or three additional applications. Nine days’ difference for all four applications to have been applied, at this stage of fruit development, may be quite significant.

Again, attempts to count the number of epidermal cells on cuticles from successive sampling dates by examining the cutin imprints became more difficult (data not shown). With each successive sample (monthly intervals), the cutin surrounding the cells became increasingly thicker and multilayered (Fig. 6). By 10 Sept., the enzymatically isolated cutin was at least three cell layers deep in places (Fig. 6D), which precluded accurate area measurements of the epidermal cell layer. Possibly, this multilayering is a response to arid conditions typical of those found in central Washington State and other hot, dry regions. Conditions of high humidity during the last month of fruit development tend to reduce cuticle thickness in such cultivars as Gala and Golden Delicious (E. Curry, personal observation). Thus, it is not surprising that fruit exposed to conditions of high desiccation stress during the final month of development, which, in this location, is often the hottest month of the year, would tend to develop a epicuticular layer optimized to reduce water loss both pre- and postharvest.

Fig. 6.
Fig. 6.

Scanning electron micrographs of the interior surface (flesh side) of representative sections of untreated ‘Golden Delicious’ apple fruit cuticle sampled on 12 June (A), 16 July (B), 13 Aug. (C), and 10 Sept. (D) and enzymatically treated to remove cell debris. Note thickening and multilayered cutin on successive sample dates. Bar is 20 μM.

Citation: HortScience horts 47, 2; 10.21273/HORTSCI.47.2.232

This also may raise concern regarding effect of overhead cooling on cuticle thickness of certain cultivars and the resultant water loss during storage. It stands to reason that fruit grown in an arid environment should be the most well protected against water loss during fruit development and, therefore, during storage where subfreezing fan coil temperatures continually extract ambient water vapor from the room. In contrast, fruit developing under conditions of high humidity, precipitation, or prolonged overhead cooling could develop a thinner cuticle less likely to protect adequately against fruit water loss, especially if the fruit were harvested during or shortly after such moisture events. Preliminary observations in which portions of individual fruit peel were subjected to high humidity (greater than 95%) during the last 4 weeks of development suggest humid microenvironments of individual fruits (e.g., leaf-to-fruit or fruit-to-fruit contact areas) may modify thickness of the newly expanded cuticle matrix, thereby weakening its resistance to desiccation stress (E. Curry, personal observations). Fruit growth rate, ambient humidity, and temperature during this period would likely contribute to the magnitude of the effects.

Gibberellins have been shown to increase cell division in different species depending on cell location and stage of development (Bangerth and Schröder, 1994; Bradley and Crane, 1957; Sachs and Lang, 1957; Wareing, 1958). Histological study on Japanese pear revealed that parthenocarpic fruits induced by GA4 and GA7 had increased cell numbers, but smaller cell size, relative to pollinated fruits (Tsujikawa et al., 1990). As Knoche et al. (2011) suggested, an increase in the planar cell density implies a structurally stronger cuticle. This is especially true if the cuticle surrounding smaller cells is similar to that of their larger counterparts. That is, if treatment with GA4+7 induces cells with smaller planar surface area while maintaining the cuticle membrane at or above thickness levels surrounding untreated cells, it stands to reason that planar surface desiccation stress per cell would also be less. Gibberellins have been reported to increase thickness of cuticle membranes isolated from rice (Oryza sativa L.) internodes (Hoffmann-Benning and Kende 1994) as well as overall weight of cuticles isolated from pea (Pisum sativum) stems (Bowen and Walton 1988) and developing tomato (Lycopersicon esculentum L.) fruit (Knoche and Peschel, 2007). More recently, Knoche et al. (2011) reported no effect of GA4+7 on weight of isolated cuticles in ‘Golden Delicious’; however, cutin is not confined to the epidermal cell surface alone; rather, it may develop around hypodermal cell layers as fruit enlarge (Fig. 6). Thus, weight of isolated cuticles may not be the best variable with which to measure the effect of GA4+7 on epidermal cuticle thickness in apples.

In pomological science texts, it is often assumed that the duration of cell division in apple fruitlets is 4 to 5 weeks after anthesis, after which fruit growth is primarily a function of cell enlargement (Westwood, 1995). My contention is the period at which “cell division ceases” identifies the developmental stage at which the majority of cortex cells simply enlarge and the epidermal planar cell area is maintained (within certain limits). That is, the planar cell area and, therefore, the epicuticular thickness are optimized according to ambient conditions. Obviously, epidermal cells do not simply enlarge after 5 weeks of fruitlet development. Were this true, epidermal cell planar area when the fruit is 80 mm in diameter would be 16 times that when the fruit was 20 mm in diameter, which, according to the calculations in Figure 5, would be ≈3200 μM2, or roughly equal to a circle with a diameter of 64 μM. Rather, it appears the cutin polymer, so protective of the epidermal cell, begins to develop around the second, third, and fourth layers of cells (hypodermis), as desiccation stress dictates. Although it is difficult to measure epidermal cell planar area through this tissue at the end of the growing season because of the obstructing cutin multilayers, the plates in Figure 6 suggest this is the case. Whether the smaller cell size induced by the GA4+7 treatments is maintained through fruit enlargement is not known.

With smaller epidermal cells, one would also expect microcracks to be thinner, shorter in length, and perhaps less invasive as suggested by the images in Figure 7C–D assuming that microcracking is mainly a function of epidermal cell division and/or sub-epidermal cell enlargement.

Fig. 7.
Fig. 7.

Effect of four treatment applications of gibberellin A4+7 at 30 mg·L−1 (B, D, F) vs. water only (A, C, E) on ‘Golden Delicious’ apple calyx-end russet (A–B), cuticular microcracks (C–D), and epidermal cell size (E–F) in 2008. Images of whole fruit calyx end russeting (A–B) were from fruit sampled on 10 Sept. Scanning electron micrographs of epicuticular microcracking (C–D) and epidermal cell cutin imprints (E–F) were from fruit sampled on 12 June. Dashed lines in E and F are yellow enhancements of grayscale overlays representative of individual measured epidermal cell planar areas.

Citation: HortScience horts 47, 2; 10.21273/HORTSCI.47.2.232

Generally, environmentally based russet development is either a direct response of the fruit epidermal cell to desiccation or an indirect response to excessive moisture. Response to desiccation may occur by: 1) physical damage resulting in exposure of cells to air with less than 100% humidity; 2) injury of epidermal cells resulting in arrested cuticle development; or 3) disruption of the protective wax layer resulting in a less than optimized water vapor barrier for given conditions. Excessive moisture, in contrast, may elicit russeting by one of two mechanisms. Standing water may increase imbibition and induce enlargement of subcuticular cells, thereby increasing cuticle microcracking against which the cell must protect. Alternatively, excessive moisture may, temporarily, deacclimate epidermal tissue. For example, when a fruitlet develops in a cool, moist environment, the fruit surface may sense little desiccation stress and, therefore, develop a cuticle optimized for such conditions. If the environment quickly becomes hot and/or arid, the cuticle suddenly is inadequate to manage the loss of water vapor, thereby resulting in “emergency management” of water loss and development of additional suberin. Russeting within the stem bowl, as another example, may be a response of deacclimated peel tissue to a cycle of evaporating moisture and increasing aridity, a response to changing conditions rather than the conditions themselves. Although there are data suggesting apples enclosed in plastic bags (100% humidity) for the entirety of the growing season (Tukey, 1959) develop russet, similar preliminary experiments in arid environments using water-impermeable but breathable resin bags have not been so conclusive (E. Curry, personal observation).

Conclusions

Data from this 2-year study using fruit from an historically high-russetting ‘Golden Delicious’ orchard suggest GA4+7 reduces environmental peel russeting by reducing epidermal cell size. Transillumination microscopy did not permit accurate measurement of ‘Golden Delicious’ fruit epidermal cell planar area beyond 6–8 weeks after anthesis because isolated cuticles became thicker and multilayered causing chromatic aberrations. On the other hand, evaluating epidermal cell planar measurements using SEM was clear and more accurate. Further work to examine the effect of localized, high-humidity microenvironments on cuticle thickness and composition as well as sub-epidermal cell organization may provide insight into postharvest disorder development and quality loss in storage.

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  • Eccher, T. & Boffelli, G. 1978 Riduzione della rugginositá epidermica della mela Golden Delicious con trattamenti di gibberelline Riv. Ortoflorofruttic. Ital. 62 205 211

    • Search Google Scholar
    • Export Citation
  • Eccher, T., Hajnajari, H., Di Lella, S. & Elli, A. 2008 Gibberellin content of apple fruit as affected by genetic and environmental factors Acta Hort. 774 221 224

    • Search Google Scholar
    • Export Citation
  • Edgerton, L.J. & Veinbrants, N. 1979 Reduction in russeting of ‘Golden Delicious’ apples with silicon dioxide formulations and gibberellins A4+7 HortScience 14 40 41

    • Search Google Scholar
    • Export Citation
  • Elfving, D.C. & Allen, O.B. 1987 Effect of gibberellin A4+7 applications on Golden Delicious fruit russet Crop Res. 27 11 18

  • Faust, M. & Shear, C.B. 1972a Russeting of apples, an interpretive review HortScience 7 233 235

  • Faust, M. & Shear, C.B. 1972b Fine structure of the fruit surface of three apple cultivars J. Amer. Soc. Hort. Sci. 97 351 355

  • Fogelman, E., Redel, G., Doron, I., Naor, A., Ben-Yashar, E. & Ginzberg, I. 2009 Control of apple russetting in a warm and dry climate J. Hort. Sci. Biotechnol. 84 279 284

    • Search Google Scholar
    • Export Citation
  • Hatch, A.H. 1975 The influence of mineral nutrition and fungicides on russeting of ‘Goldspur' apple fruit J. Amer. Soc. Hort. Sci. 100 52 55

  • Heidenreich, M.C.M., Corral-Garcia, M.R., Momol, E.A. & Burr, T.J. 1997 Russet of apple fruit caused by Aureobasidium pullulans Plant Dis. 81 337 342

  • Hoffmann-Benning, S. & Kende, H. 1994 Cuticle biosynthesis in rapidly growing internodes of deepwater rice Plant Physiol. 104 719 723

  • Ju, Z. & Bramlage, W.J. 1999 Phenolics and lipid-soluble antioxidants in fruit cuticle of apples and their antioxidant activities in model systems Postharvest Biol. Technol. 16 107 118

    • Search Google Scholar
    • Export Citation
  • Knoche, M. & Grimm, E. 2008 Surface moisture induces microcracks in the cuticle of ‘Golden Delicious’ apple HortScience 43 1929 1931

  • Knoche, M., Khanal, B. & Stopar, M. 2011 Russeting and microcracking of ‘Golden Delicious’ apple fruit concomitantly decline due to gibberellin A4+7 application J. Amer. Soc. Hort. Sci. 136 159 164

    • Search Google Scholar
    • Export Citation
  • Knoche, M. & Peschel, S. 2007 Gibberellins increase cuticle deposition in developing tomato fruit Plant Growth Regulat. 51 1 10

  • Lallu, N., Haynes, G., Pidakala, P., Billing, D., Johnston, J., Francis, K. & McDermott, K. 2010 Factors affecting the incidence of ‘russet browning' disorder in’ Cox's Orange Pippin' apples Acta Hort. 877 499 506

    • Search Google Scholar
    • Export Citation
  • Lichstein, H.C. & Soule, M.H. 1943 Studies of the effect of sodium azide on microbic growth and respiration J. Bacteriol. 47 221 230

  • Lindow, S.E., Desurmont, C., Elkins, R., McGourty, G., Clark, E. & Brandl, M.T. 1998 Occurrence of indole-3-acetic acid-producing bacteria on pear trees and their association with fruit russet Phytopathology 88 1149 1157

    • Search Google Scholar
    • Export Citation
  • McCormick, R. & Streif, J. 2008 Stem end russet browning of ‘Cox's orange pippin’ apples, an undesired side effect from the application of 1-MCP Acta Hort. 796 125 128

    • Search Google Scholar
    • Export Citation
  • Meador, D.B. & Taylor, B.H. 1987 Effect of early season foliar sprays of GA4+7 on russeting and return bloom of ‘Golden Delicious’ apple HortScience 22 412 415

    • Search Google Scholar
    • Export Citation
  • Meyer, A. 1944 A study of the skin structure of Golden Delicious apples. Proc. Amer. Soc. Hort. Sci. 45:105–110.

  • Opara, L.U., Studman, C.J. & Banks, N.H. 1997 Fruit skin splitting and cracking, p. 217–262. In: Janick, J. (ed.). Horticultural reviews. Vol. 19. John Wiley & Sons, Inc., Oxford, U.K.

  • Sachs, R.M. & Lang, A. 1957 Effect of gibberellin on cell division in Hyoscyamus Science 125 1144 1145

  • Sanchez, E.E., Righetti, T.L., Sugar, D. & Lombard, P.B. 1992 Effects of timing of nitrogen application on nitrogen partitioning between vegetative, reproductive, and structural components of mature ‘Comice’ pears J. Hort. Sci. 67 51 58

    • Search Google Scholar
    • Export Citation
  • Skene, D.S. & Greene, D.W. 1982 The development of russet, rough russet and cracks on the fruit of the apple ‘Cox's Orange Pippin’ during the course of the season J. Hort. Sci. 57 165 174

    • Search Google Scholar
    • Export Citation
  • Steenkamp, J. & Westraad, I. 1988 Effect of gibberellin A4+7 on stem- and calyx-end russeting in ‘Golden Delicious’ apples Sci. Hort. 35 207 215

    • Search Google Scholar
    • Export Citation
  • Taylor, B.K. 1975 Reduction of apple skin russeting by gibberellin A4+7 J. Hort. Sci. 50 169 172

  • Tsujikawa, T., Ichii, T., Nakanishi, T., Ozaki, T. & Kawai, Y. 1990 In vitro flowering of Japanese pear and the effect of GA4+7 Sci. Hort. 41 233 245

  • Tukey, L.D. 1959 Observations on the russeting of apples growing in plastic bags J. Amer. Soc. Hort. Sci. 74 30 39

  • Wareing, P.F. 1958 Interaction between indoleacetic acid and gibberellic acid in cambial activity Nature 181 1744 1745

  • Westwood, M.N. 1995 Temperate-zone pomology, physiology and culture. 3rd Ed. Timber Press, Portland, OR.

  • Wood, G.A. 2010 Sensitivity of apple (Malus domestica) indicator cultivars to russet ring disease, and the results of graft—Transmission trials of other fruit—Affecting disorders of apple N. Z. J. Crop Hort. Sci. 29 255 265

    • Search Google Scholar
    • Export Citation

Contributor Notes

Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

To whom reprint requests should be addressed; e-mail eric.curry@ars.usda.gov.

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    Gray-scale image of the green channel (RGB) from a representative brightfield TIFF image similar to those from which planar epidermal cell density was determined for gibberellin A4+7 treatments on ‘Golden Delicious’ apple in 2007. Dotted lines indicate quadrant from which number of cells was counted. Bar is 10 μM.

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    Graph of planar cell density vs. treatment application rate (two independent observers) from ‘Golden Delicious’ apple epidermal tissue sampled on 8 June 2007 and enzymatically treated to remove cell debris. Bars indicate + se.

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    Scanning electron micrograph of the interior surface (flesh side) of a representative section of untreated ‘Golden Delicious’ apple fruit cuticle sampled on 12 June 2008 and enzymatically treated to remove cell debris. Example of individual cell area measurement outlines are in black. Arrow indicates developing cutin from recent cell division. Bar is 10 μM.

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    Graph of planar cell area and planar cell density vs. treatment application rate from ‘Golden Delicious’ apple epidermal tissue sampled on 12 June 2008 and enzymatically treated to remove cell debris. Scanning electron micrographs were used for measurements. Bars indicate + se.

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    Graph of planar cell surface area vs. fruitlet diameter of untreated ‘Golden Delicious’ apple cuticles isolated during the first ≈5 weeks of fruitlet development. Inset scanning electron micrographs indicate representative cutin imprints of epidermal cells from which measurements were made. Bars indicate ± se.

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    Scanning electron micrographs of the interior surface (flesh side) of representative sections of untreated ‘Golden Delicious’ apple fruit cuticle sampled on 12 June (A), 16 July (B), 13 Aug. (C), and 10 Sept. (D) and enzymatically treated to remove cell debris. Note thickening and multilayered cutin on successive sample dates. Bar is 20 μM.

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    Effect of four treatment applications of gibberellin A4+7 at 30 mg·L−1 (B, D, F) vs. water only (A, C, E) on ‘Golden Delicious’ apple calyx-end russet (A–B), cuticular microcracks (C–D), and epidermal cell size (E–F) in 2008. Images of whole fruit calyx end russeting (A–B) were from fruit sampled on 10 Sept. Scanning electron micrographs of epicuticular microcracking (C–D) and epidermal cell cutin imprints (E–F) were from fruit sampled on 12 June. Dashed lines in E and F are yellow enhancements of grayscale overlays representative of individual measured epidermal cell planar areas.

  • Bangerth, F. & Schröder, M. 1994 Strong synergistic effects of gibberellins with the synthetic cytokinin N-(2-chloro-4-pyridyl)-N-phenylurea on parthenocarpic fruit set and some other fruit characteristics of apple Plant Growth Regulat. 15 293 302

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  • Bowen, D.J. & Walton, T.J. 1988 Cutin composition and biosynthesis during gibberellic-acid induced stem extension of Pisum sativum var Meteor. Plant Sci 55 115 127

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    • Export Citation
  • Bradley, M.V. & Crane, J.C. 1957 Gibberellin-stimulated cambial activity in stems of apricot spur shoots Science 126 972 973

  • Bukovac, M.J. & Nakagawa, S. 1968 Gibberellin-induced asymmetric growth of apple fruits HortScience 3 172 173

  • Creasy, L.L. 1980 The correlation of weather parameters with russet of ‘Golden Delicious’ apples under orchard conditions J. Amer. Soc. Hort. Sci. 105 735 738

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  • Creasy, L.L. & Swartz, H.J. 1981 Agents influencing russet on ‘Golden Delicious’ apple fruits J. Amer. Soc. Hort. Sci. 106 203 206

  • Curry, E.A. 2005 Ultrastructure of epicuticular wax aggregates during fruit development in apple (Malus domestica Borkh.) J. Hort. Sci. Biotechnol. 80 668 676

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  • Daines, R., Weber, D.J., Bunderson, E.D. & Roper, T. 1984 Effect of early sprays on control of powdery mildew fruit russet on apples Plant Dis. 68 326 328

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  • Duso, C., Castagnoli, M., Simoni, S. & Angeli, G. 2010 The impact of eriophyoids on crops: Recent issues on aculus schlechtendali, calepitrimerus vitis and aculops lycopersici Exp. Appl. Acarol. 51 151 168

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  • Easterbrook, M.A. & Fuller, M.M. 1986 Russeting of apples caused by apple rust mite Aculus schlechtendali Acarina Eriophyidae Ann. Appl. Biol. 109 1 10

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    • Export Citation
  • Eccher, T. 1975 Influenza di alcuni fitormoni sulla rugginosita della ‘Golden Delicious’ Rivista dell'Ortoflorofrutticoltura Italiana 59 246 261

    • Search Google Scholar
    • Export Citation
  • Eccher, T. & Boffelli, G. 1978 Riduzione della rugginositá epidermica della mela Golden Delicious con trattamenti di gibberelline Riv. Ortoflorofruttic. Ital. 62 205 211

    • Search Google Scholar
    • Export Citation
  • Eccher, T., Hajnajari, H., Di Lella, S. & Elli, A. 2008 Gibberellin content of apple fruit as affected by genetic and environmental factors Acta Hort. 774 221 224

    • Search Google Scholar
    • Export Citation
  • Edgerton, L.J. & Veinbrants, N. 1979 Reduction in russeting of ‘Golden Delicious’ apples with silicon dioxide formulations and gibberellins A4+7 HortScience 14 40 41

    • Search Google Scholar
    • Export Citation
  • Elfving, D.C. & Allen, O.B. 1987 Effect of gibberellin A4+7 applications on Golden Delicious fruit russet Crop Res. 27 11 18

  • Faust, M. & Shear, C.B. 1972a Russeting of apples, an interpretive review HortScience 7 233 235

  • Faust, M. & Shear, C.B. 1972b Fine structure of the fruit surface of three apple cultivars J. Amer. Soc. Hort. Sci. 97 351 355

  • Fogelman, E., Redel, G., Doron, I., Naor, A., Ben-Yashar, E. & Ginzberg, I. 2009 Control of apple russetting in a warm and dry climate J. Hort. Sci. Biotechnol. 84 279 284

    • Search Google Scholar
    • Export Citation
  • Hatch, A.H. 1975 The influence of mineral nutrition and fungicides on russeting of ‘Goldspur' apple fruit J. Amer. Soc. Hort. Sci. 100 52 55

  • Heidenreich, M.C.M., Corral-Garcia, M.R., Momol, E.A. & Burr, T.J. 1997 Russet of apple fruit caused by Aureobasidium pullulans Plant Dis. 81 337 342

  • Hoffmann-Benning, S. & Kende, H. 1994 Cuticle biosynthesis in rapidly growing internodes of deepwater rice Plant Physiol. 104 719 723

  • Ju, Z. & Bramlage, W.J. 1999 Phenolics and lipid-soluble antioxidants in fruit cuticle of apples and their antioxidant activities in model systems Postharvest Biol. Technol. 16 107 118

    • Search Google Scholar
    • Export Citation
  • Knoche, M. & Grimm, E. 2008 Surface moisture induces microcracks in the cuticle of ‘Golden Delicious’ apple HortScience 43 1929 1931

  • Knoche, M., Khanal, B. & Stopar, M. 2011 Russeting and microcracking of ‘Golden Delicious’ apple fruit concomitantly decline due to gibberellin A4+7 application J. Amer. Soc. Hort. Sci. 136 159 164

    • Search Google Scholar
    • Export Citation
  • Knoche, M. & Peschel, S. 2007 Gibberellins increase cuticle deposition in developing tomato fruit Plant Growth Regulat. 51 1 10

  • Lallu, N., Haynes, G., Pidakala, P., Billing, D., Johnston, J., Francis, K. & McDermott, K. 2010 Factors affecting the incidence of ‘russet browning' disorder in’ Cox's Orange Pippin' apples Acta Hort. 877 499 506

    • Search Google Scholar
    • Export Citation
  • Lichstein, H.C. & Soule, M.H. 1943 Studies of the effect of sodium azide on microbic growth and respiration J. Bacteriol. 47 221 230

  • Lindow, S.E., Desurmont, C., Elkins, R., McGourty, G., Clark, E. & Brandl, M.T. 1998 Occurrence of indole-3-acetic acid-producing bacteria on pear trees and their association with fruit russet Phytopathology 88 1149 1157

    • Search Google Scholar
    • Export Citation
  • McCormick, R. & Streif, J. 2008 Stem end russet browning of ‘Cox's orange pippin’ apples, an undesired side effect from the application of 1-MCP Acta Hort. 796 125 128

    • Search Google Scholar
    • Export Citation
  • Meador, D.B. & Taylor, B.H. 1987 Effect of early season foliar sprays of GA4+7 on russeting and return bloom of ‘Golden Delicious’ apple HortScience 22 412 415

    • Search Google Scholar
    • Export Citation
  • Meyer, A. 1944 A study of the skin structure of Golden Delicious apples. Proc. Amer. Soc. Hort. Sci. 45:105–110.

  • Opara, L.U., Studman, C.J. & Banks, N.H. 1997 Fruit skin splitting and cracking, p. 217–262. In: Janick, J. (ed.). Horticultural reviews. Vol. 19. John Wiley & Sons, Inc., Oxford, U.K.

  • Sachs, R.M. & Lang, A. 1957 Effect of gibberellin on cell division in Hyoscyamus Science 125 1144 1145

  • Sanchez, E.E., Righetti, T.L., Sugar, D. & Lombard, P.B. 1992 Effects of timing of nitrogen application on nitrogen partitioning between vegetative, reproductive, and structural components of mature ‘Comice’ pears J. Hort. Sci. 67 51 58

    • Search Google Scholar
    • Export Citation
  • Skene, D.S. & Greene, D.W. 1982 The development of russet, rough russet and cracks on the fruit of the apple ‘Cox's Orange Pippin’ during the course of the season J. Hort. Sci. 57 165 174

    • Search Google Scholar
    • Export Citation
  • Steenkamp, J. & Westraad, I. 1988 Effect of gibberellin A4+7 on stem- and calyx-end russeting in ‘Golden Delicious’ apples Sci. Hort. 35 207 215

    • Search Google Scholar
    • Export Citation
  • Taylor, B.K. 1975 Reduction of apple skin russeting by gibberellin A4+7 J. Hort. Sci. 50 169 172

  • Tsujikawa, T., Ichii, T., Nakanishi, T., Ozaki, T. & Kawai, Y. 1990 In vitro flowering of Japanese pear and the effect of GA4+7 Sci. Hort. 41 233 245

  • Tukey, L.D. 1959 Observations on the russeting of apples growing in plastic bags J. Amer. Soc. Hort. Sci. 74 30 39

  • Wareing, P.F. 1958 Interaction between indoleacetic acid and gibberellic acid in cambial activity Nature 181 1744 1745

  • Westwood, M.N. 1995 Temperate-zone pomology, physiology and culture. 3rd Ed. Timber Press, Portland, OR.

  • Wood, G.A. 2010 Sensitivity of apple (Malus domestica) indicator cultivars to russet ring disease, and the results of graft—Transmission trials of other fruit—Affecting disorders of apple N. Z. J. Crop Hort. Sci. 29 255 265

    • Search Google Scholar
    • Export Citation
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