Effects of Promalin on Fruit Growth and Cuticle Properties of ‘Pinova’ Apple

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Bishnu P. Khanal Institute of Horticultural Production Systems, Fruit Science Section, Leibniz University Hannover, Herrenhäuser Straβe 2, 30419 Hannover, Germany

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Prabin Dahal Institute of Horticultural Production Systems, Fruit Science Section, Leibniz University Hannover, Herrenhäuser Straβe 2, 30419 Hannover, Germany

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Yiru Si Institute of Horticultural Production Systems, Fruit Science Section, Leibniz University Hannover, Herrenhäuser Straβe 2, 30419 Hannover, Germany

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Moritz Knoche Institute of Horticultural Production Systems, Fruit Science Section, Leibniz University Hannover, Herrenhäuser Straβe 2, 30419 Hannover, Germany

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Oliver K. Schlüter Department of System Process Engineering, Leibniz Institute for Agricultural Engineering and Bioeconomy (ATB), Max-Eyth-Allee 100, 14469 Potsdam, Germany

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Abstract

Promalin (Valent BioSciences, Libertyville, IL, USA) is a proprietary mixture of gibberellin A4 + 7 and 6-benzyladenine that is widely used in apple production to improve the fruit shape, size, and skin quality. Promalin typically increases fruit size and length. However, the increased growth likely increases the strain in the fruit skin, which may exacerbate microcracking of the cuticle and, consequently, russeting. This study aimed to monitor the growth-stimulating effect of Promalin in three different regions of fruits and investigate whether Promalin affects cuticular microcracking via effects on the deposition of cuticular components or via the accumulation of strain in the cuticle. Four Promalin sprays (20 mg⋅L1) were applied to runoff; the first was applied at full bloom, and the remaining sprays were applied at approximately weekly intervals thereafter. Fruit surface areas and fruit surface area growth rates of the Promalin-treated fruits were higher than those of the untreated control fruits. Promalin increased the fruit length, but it had no effect on the fruit equatorial diameter. In Promalin-treated fruits, the base of each sepal extended, thickened, and became fleshy as early as 15 days after full bloom (DAFB). Allometric growth analyses revealed higher constant differential growth ratios of the pedicel and calyx length (before 36 DAFB) in Promalin-treated fruits than in control fruits. After 36 DAFB, the difference in constant differential growth ratios between Promalin-treated fruits and control fruits decreased. Cuticle mass per unit area increased with time in all regions of the fruit surface and was slightly (+3.3%) but significantly higher in fruits treated with Promalin than in control fruits. Additionally, the biaxial strain release was slightly and significantly lower in Promalin-treated fruits than in control fruits. When the isolated, cuticle was ablated from the inner surface and dewaxed, strain relaxation in the control fruits was higher than that in the Promalin-treated fruits. It was concluded that Promalin treatment increases the length of the fruit by increasing the lengths of the pedicel and calyx regions early during fruit development. Promalin only slightly increased cuticle deposition and fixation of cuticular strain. Promalin had no effects on microcracking or russeting.

Promalin (Valent BioSciences, Libertyville, IL, USA) is a plant growth regulator mix that is used quite commonly in commercial apple production. It comprises gibberellins A4 and A7 (GA4 + 7), which stimulate cell division and increase the number of epidermal cells per unit of fruit surface area, and cytokinin 6-benzyladenine (6-BA) (Curry 2012). In apple, GA4 + 7 increases the structural support of the cuticle and, thus, the strength of the fruit skin. It also reduces microcracking and russeting (Curry 2012; Eccher and Boffelli 1981; Knoche et al. 2011). However, 6-BA stimulates cell division and differentiation in apple fruit (Ginzberg and Stern 2016).

Promalin is reported to improve fruit shape, fruit size, and visual skin quality in apples. Early-season Promalin applications extend the fruit length, thus increasing the length-to-diameter ratio (Curry and Williams 1983; Veinbrants and Miller 1981). Interestingly, Promalin also decreases russeting (Leite et al. 2006). This is somewhat surprising because russeting results from the formation of microscopic cracks in the cuticle. Microscopic cracks, in turn, result from excessive strain of the cuticle. This study included two hypotheses. Hypothesis 1 comprised the following: one could reasonably expect that the higher growth rate of Promalin-treated fruits and, hence, slightly larger size of fruits to result in higher skin strain, more cuticular microcracking, and more russeting. For example, the calyx and cheek regions of russet-susceptible pear cultivars have higher growth rates than the neck regions and are consequently more russeted (Scharwies et al. 2014). However, hypothesis 2 comprised the following: it could be argued that any increases in cutin and/or wax deposition in response to Promalin treatment could offset the effects of the increased growth on strain, microcracking, and russeting. This is because increased cutin and/or wax deposition are known to fix elastic strain in the cuticle by converting elastic into plastic strain, thus making microcracking less likely (Khanal et al. 2013, 2014; Si et al. 2021). The objective of this study was to investigate these two hypotheses. We studied the effects of Promalin on fruit growth, cuticle deposition, and cuticular strain relaxation of apple fruits and used untreated fruits as controls.

Materials and Methods

Plant materials and treatments.

The apple cultivar Pinova (Malus ×domestica Borkh.) was used during this study. The trees were grafted on M9 rootstocks and grown in an experimental orchard of the Leibniz University of Hanover in Ruthe, Germany (52°14′N, 9°49′E). The experiment comprised a total of 50 uniform trees within a single row. Of these, 25 trees were sprayed four times to runoff with Promalin at 20 mg⋅L−1 according to the manufacturer’s specifications (Valent BioSciences). Applications were performed at full bloom, 8 d after full bloom (DAFB), 15 DAFB, and 26 DAFB. The remaining 25 trees were sprayed with water and served as controls. The individual trees sprayed with Promalin (treated) and those sprayed with water (controls) were randomly distributed within the same row.

Fruit growth.

Fruit growth was monitored non-destructively. Uniform fruits were selected and tagged. Calibrated images of the fruits (still attached) were obtained at 1- to 2-week intervals between 6 DAFB and maturity. The application of Promalin alters the fruit shape (Curry and Williams 1983; Veinbrants and Miller 1981). Thus, the Promalin-treated fruits were slightly elongated, whereas the control fruits were more spherical. To quantify the surface area of Promalin-treated fruits, they were cut (virtually) into three to six thick transverse sections. Each section was considered to be a truncated cone. For each section, the maximum radius (R), minimum radius (r), and length (l) were quantified using image analysis software (CellP; Olympus, Hamburg, Germany). The following equation was used to calculated the lateral skin surface area (S) of each section:
S = π(R + r)(Rr)2+l2
For the most proximal (pedicel end) and the most distal (calyx end) sections, the top and bottom were assumed to represent a flat area of a circular shape and calculated as the areas of two circles. The skin surface areas of all sections were summed to yield the total surface area (A) of a Promalin-treated apple fruit. The model was calibrated by peeling the fruit and measuring the surface area of the peel spread on a glass plate. A highly significant relationship was observed between the fruit surface area predicted using the aforementioned model and that measured using the excised peel using the following equation:
(Areacalc = 0.90(±0.01) × Areameas, r2 = 0.99***).

For the control fruits, the maximum length and two orthogonal equatorial diameters were quantified by image analysis. Assuming sphericity, the total fruit surface area (A) was calculated from the mean of the length and the two diameters.

The change in the surface area with time was described using a sigmoidal regression model. The growth rate of surface area was calculated from the first derivative of this regression model. Relative surface area growth rates based on the whole fruit at any one time were calculated by dividing the surface area growth rate by the absolute fruit surface area at that time.

Allometric growth of different regions of the fruits.

Fruits were sampled at 1- to 2-week intervals starting from full bloom until maturity and cut longitudinally into two halves. Calibrated images of the cross-sectional surface were prepared using a binocular microscope (MZ6; Leica Microsystems, Wetzlar, Germany) or digital camera (Canon EOS 550D, EF-S 18–55 mm; Canon, Tokyo, Japan) mounted on a photo stand. Subsequently, the following dimensions were measured using image analysis (CellP): diameter at the base of the core (referred to as the diameter of the pedicel region), center diameter of the fruit, diameter at the top of the core (referred to as the diameter of the calyx region), core diameter (the ovary portion), total length, length at the base of the core (pedicel region length), core length, and length at the top of the core (calyx region length) (Fig. 3).

An allometric growth analysis was performed as described previously (Khanal et al. 2023; Skene 1966). Briefly, the log10 of all diameters and lengths were calculated and plotted against the log10 of the center diameter (Skene 1966). The slopes of these log–log relationships represent the rates of growth of each particular region of the fruit relative to the growth of the diameter of the center region of the fruit. This slope term is referred to as the “constant differential growth ratio,” as suggested by Huxley (1924). A constant differential growth ratio of 1 indicates that the growth rate in a particular region of the fruit is the same as that in the center region (i.e., growth is isoallometric). A constant differential growth ratio >1 implies that the growth rate in a particular region of the fruit is faster (“hyperallometric”) than that in the center region. If the constant differential growth ratio is <1, then the growth is slower (hypoallometric) than that in the center region.

Cuticle isolation and wax extraction.

Cuticles were isolated from fruits harvested from 32 DAFB and onward. Earlier samplings were not practicable because of the small size of younger fruitlets. Excised skin segments comprising the cuticle, epidermis, hypodermis, and some adhering flesh were prepared from the pedicel region, center region, and calyx region using a biopsy punch (6-mm or 8-mm diameter; Kai Europe, Solingen, Germany). The excised skin segments were incubated in a solution containing pectinase (90 mL⋅L−1; Panzym Super E flüssig, Novozymes A/S, Krogshoejvej, Bagsvaerd, Denmark) and cellulase (5 mL⋅L−1; cellubrix L; Novozymes A/S). The enzyme solution was buffered using 50 mm citric acid buffer. The pH was adjusted to 4.0 using NaOH. Sodium azide (NaN3) was added at a final concentration of 30 mm to prevent microbial growth. The solution was refreshed two to five times until the cuticular membranes (CMs) separated from the adhering tissue. The isolated CMs were thoroughly rinsed and cleaned in deionized water using a soft brush. Thereafter, the CMs were dried and maintained at room temperature until use. Cuticular wax was extracted in a Soxhlet apparatus using CHCl3/methanol (1:1 v/v) for approximately 2 h (minimum of three extraction cycles). The masses of CM and dewaxed CM (DCM) discs were determined gravimetrically on a microbalance (CPA2P; Sartorius, Göttingen, Germany). The mass per unit area was calculated by dividing by the cross-sectional area of the biopsy punch. The number of replications was six, with one replication comprising 10 CM or DCM discs.

Strain relaxation analysis of the cuticle.

A strain relaxation analysis was performed according to the procedure described in detail by Lai et al. (2016). Briefly, the skin segment discs (6-mm or 8-mm diameter) were excised from the pedicel, equatorial, and calyx regions of the fruits. The CMs were isolated enzymatically as described previously. Subsequently, the CMs were cleaned and spread on a microscope slide. Calibrated images of the CM discs were obtained using a binocular microscope (Wild M10 [Leica Microsystem] and camera DP71 [Olympus]). Thereafter, CMs were dried, dewaxed, rehydrated for 12 h, and photographed again. Two orthogonal diameters per CM or DCM disc were measured using an image analysis (CellP). The areas of the CM (ACM) or DCM (ADCM) disc were calculated from the mean of the two orthogonal diameters of the CM or DCM disc. The apparent strain releases on excision of the excised skin segments and on isolation of the CM (ɛexc+iso) and the apparent strain release on wax extraction (ɛextr) were quantified using the following equations:
εexc+iso(%) = (AiACMADCM)100
εextr(%) = (ACMADCMADCM)100

In the aforementioned equations, Ai represents the area of the skin segments before excision, which equaled the cross-sectional area of the biopsy punch used to excise the skin segments. The total apparent strain release of the cuticle (ɛtot) equaled the sum of ɛexc+iso and ɛextr. The number of replications was 20.

Cold atmospheric pressure plasma treatment.

The deposition of cutin occurs on the morphological inner side of the apple fruit cuticle (Si et al. 2021). The cutin deposited on a strained cuticle “fixes” the elastic strain in the CM by converting elastic reversible strain into plastic irreversible strain (Khanal et al. 2014). Therefore, any putative effect of Promalin on cuticle deposition could alter strain fixation and, hence, strain relaxation of the cuticle upon isolation and wax extraction. To investigate this hypothesis, a strain relaxation analysis was performed following progressive ablations of the cuticle from its inner surface using cold atmospheric pressure plasma (CAPP). This ablation occurs under ambient temperature conditions without any significant heating of the cuticle (Khanal et al. 2014).

The CAPP treatment was performed following the procedure established previously (Khanal et al. 2014; Si et al. 2021). Briefly, a square pattern of four holes (sides: approximately 2.8 × 2.8 mm; hole diameters: approximately 0.5 mm) was punched in the center of a fully hydrated, isolated CM disc (12-mm diameter) using a custom-made punch. The purpose of the hole pattern was to facilitate the measurement of strain release after wax extraction (for details, see Lai et al. 2016). When wax is extracted, DCMs typically curl, and it becomes difficult to flatten a whole DCM for area quantification. However, the center area of the DCM demarcated by the four holes usually remains sufficiently flat so that strain relaxation can be quantified using the square pattern of holes. Following punching, the CMs were dried overnight at 40 °C and weighed on a microbalance (CPA2P; Sartorius). Subsequently, the inner surface of the CM was subjected to CAPP treatment for 0, 5, 10, 15, 20, and 25 min. Increasing exposure to CAPP results in increasing amounts of CM being ablated (Khanal et al. 2014; Si et al. 2021). The operating conditions of the plasma jet (8 W; kINPen 09; Neoplas tools, Greifswald, Germany) were those established previously (Khanal et al. 2014; Si et al. 2021): gas, 99.9% argon plus 0.1% oxygen at 5.4 L⋅min−1; high-frequency voltage, 1.1 MHz; peak-to-peak voltage, 2–6 kV; and power supplied to the jet, 65 V with resonance balancing of 0.05 A. The plasma jet was operated at ambient temperature and pressure.

Determination of mass loss and biaxial strain release of CM after CAPP treatment.

Following ablation, mass loss of the CM, DCM, and wax and the biaxial strain release of the CM (on wax extraction) were quantified. From the CAPP-exposed surface of the CM disc, a 6-mm-diameter disc (referred to hereafter as a “core disc”) that included all four punched holes was excised using a biopsy punch. Core discs were dried in an oven at 40 °C for 12 h. Mass was quantified using a microbalance (CPA2P; Sartorius). The mass of the CM core discs before CAPP treatment was calculated from the mass of the 12-mm CM disc obtained before CAPP ablation. The mass loss of the CM during CAPP ablation was calculated as the difference in the mass of the core disc before and after CAPP ablation.

Subsequently, the CM core discs were hydrated for 12 h, and a calibrated photograph of the CM including the four punched holes was obtained using a dissecting microscope (Wild M10 [Leica Microsystem] and camera DP71 [Olympus]). The CM core discs were air-dried, dewaxed, and oven-dried at 40 °C for 12 h. Then, the mass of the DCM core discs was quantified. The mass loss of DCM and wax per unit surface area were calculated. The DCM core disc was rehydrated for 12 h, and images of the DCM including the four holes were obtained. The surface areas of the squares enclosed by the four holes in the CM (ACM (CAPP)) after CAPP treatment and in the DCM (ADCM (CAPP)) were quantified using an image analysis (CellP). The biaxial strain release of the ablated CM after wax extraction (ɛextr(CAPP)) was calculated using the following equation:
εextr(CAPP)(%) = (ACM(CAPP)ADCM(CAPP)ADCM(CAPP))100

Monitoring of microcracking.

Control and Promalin-treated fruits were sampled at 23, 32, 47, and 69 DAFB. Formation of microcracks in the cuticle of the pedicel, center, and calyx region was monitored using the fluorescent tracer acridine orange (AO; Carl Roth, Karlsruhe, Germany). Penetration of acridine orange is limited to microscopic cracks and, occasionally, to lenticels in the cuticle and does not occur through an intact cuticle. Briefly, fruits were incubated in 0.1% (w/w) AO for 10 min, removed from the solution, rinsed with deionized water, blotted using soft tissue paper, and observed under a fluorescence binocular microscope (MZ10F; Leica Microsystems). The area infiltrated by AO exhibited yellow and green fluorescence under incident fluorescence light (480–440 nm excitation; ≥510 nm emission wavelength) (Camera DP71 and GFP-plus filter; Olympus). Digital photographs were obtained and the area infiltrated was quantified by an image analysis (CellP).

Determination of water vapor permeance.

The water vapor permeance of the skin in the pedicel, center, and calyx regions of the control and Promalin-treated fruits was quantified at 69 and 147 DAFB. The skin segments were excised and the cut surface was blotted dry using soft tissue paper; then, they were mounted on stainless steel diffusion cells (Geyer and Schönherr 1988; Knoche et al. 2000) using high-vacuum grease (Korasilon-Paste; Kurt Obermeier, Bad Berleburg, Germany). The lid was sealed against the bottom of the diffusion cell using clear transparent tape (Tesa film; Beiersdorf, Norderstedt, Germany) to eliminate the potential leakage of water vapor. The diffusion cells were turned upside-down and filled with deionized water through a port in the bottom. The port was tape-sealed. The diffusion cells were equilibrated overnight; during the next morning, it was positioned in a polyethylene box on a metal grid above the dry silica gel. The amount of water lost was quantified by repeatedly weighing diffusion cells. The flow rate (F in g⋅h−1) was calculated as the slope of a linear regression line fitted through a plot of cumulative weight loss vs. time. The permeance (P; m⋅s−1) of the excised skin segments was calculated using the following equation:
P = F(A×ΔC)

In this equation, A and ΔC represent the area of the transpiring surface of the excised skin segments (m2) and the difference in water vapor concentration between the inside and outside of the diffusion cell (g⋅m−3), respectively. Because the water vapor concentration above the dry silica gel is essentially zero (Geyer and Schönherr 1988), the value of ΔC equals the water vapor concentration at saturation (20.59 g⋅m−3 at 23 °C) (Nobel 1999).

Data analysis and presentation.

Data presented in the tables and figures (except Fig. 3, where individual data points are shown) are presented as the mean ± SE. When error bars are not visible in the figures, they are smaller than the data symbols. Data were analyzed using a regression analysis or analysis of variance. Means were separated using Tukey’s Studentized range test or the Tukey-Kramer test at P ≤ 0.05. All statistical analyses were performed using the statistical software package SAS (SAS version 9.4; SAS Institute Inc., Cary, NC, USA).

Results

The increase in the fruit surface area during development of both the control and Promalin-treated fruits followed a typical sigmoid growth pattern. The surface area of the Promalin-treated fruits was consistently larger than that of the untreated control fruits (Fig. 1A). Accordingly, the rate of growth of the surface area of the Promalin-treated fruits was higher than that of the control fruits. There were no differences in relative growth rates between the treated and control fruits (Fig. 1A insets).

Fig. 1.
Fig. 1.

Developmental time course of the change in the surface area (A), length (B), and diameter (C) of the control and Promalin-treated ‘Pinova’ apple fruits. Inset in (A) upper left: Time course of change in the surface area growth rate. Inset in (A) lower right: Time course of change in the surface area relative growth rate. Inset in (B): Time course of change in the length-to-diameter ratio. Scale of x-axes in days after full bloom (DAFB). Representative images of control (D) and Promalin-treated ‘Pinova’ apple fruits (E) at maturity. Data points represent the mean ± SE. Bar = 2 cm.

Citation: HortScience 59, 7; 10.21273/HORTSCI17870-24

Promalin-treated fruits had the typical elongated shape of the calyx, whereas the untreated control fruits remained approximately spherical (Fig. 1D and 1E). The elongated shape was attributable to a consistent increase in length of the Promalin-treated fruits compared with that of the control fruits. The equatorial diameter was unaffected by the Promalin treatment (Fig. 1B and 1C).

Longitudinal cross-sections revealed that the increase in fruit length was primarily attributable to increased growth in the calyx and pedicel regions. The increased growth in the calyx region was clearly visible as early as 14 DAFB (i.e., after the second Promalin treatment). In particular, the bases of the sepals thickened and became part of the parenchymatous receptacle (Fig. 2B–E and 2H–K). By 36 DAFB, the Promalin-treated fruits exhibited the typical and well-known extension in the calyx region.

Fig. 2.
Fig. 2.

Longitudinal cross-sections of untreated control (A–F) and Promalin-treated (G–L) ‘Pinova’ apple fruits. Fruits were harvested at various stages of development. The scale bar in (A) is applicable to (A, B, D, E, H, J, and K). The scale bar in (C) applies to (C and I). The scale bar in (F) applies to (F and L). DAFB = days after full bloom.

Citation: HortScience 59, 7; 10.21273/HORTSCI17870-24

An allometric analysis revealed that the increases in the pedicel region diameter, core diameter, and calyx region diameter were all isoallometric (slopes of log–log plots were equal to or similar to 1) to the increase in the center diameter for both the Promalin-treated and control fruits (Fig. 3B–D and Table 1). In contrast, the growth in the total length and the growth in the length of the center region were hypoallometric (slope <1); i.e., slower than the increase in the center diameter) (Fig. 3E and 3G and Table 1). For both parameters, the Promalin-treated fruits had higher constant differential growth ratios than the control fruits.

Fig. 3.
Fig. 3.

(A) Sketch of the longitudinal cross-section through an apple fruit illustrating the nomenclature used to describe the different dimensions of the fruit in the calyx, center, and pedicel regions. (B–D) Relationship between the log-transformed diameters in the pedicel region (B), core (C), and calyx region (D) and log-transformed center diameter of the fruit. (E–H) Relationship between the log-transformed lengths of the whole fruit (E), pedicel region length (F), core length (G), and calyx region length (H) and log-transformed center diameter of the fruit. The slopes (constant differential growth ratios) and intercepts of these relationships are shown in Table 1.

Citation: HortScience 59, 7; 10.21273/HORTSCI17870-24

Table 1.

Regression parameters for the relationships of log-transformed diameters and lengths of the different sections of ‘Pinova’ apple fruits with the log-transformed center diameter of the fruits. Fruits were treated with Promalin. Untreated fruits served as controls.

Table 1.

It is interesting that the allometric relationships with the pedicel region length and calyx region length were biphasic. Up to 36 DAFB, the increases in both the pedicel and calyx region lengths were hypoallometric for both Promalin-treated and control fruits, and the slopes of the Promalin-treated fruits were higher than those of the controls. From 37 DAFB and onward, until maturity, the increases in the pedicel region length of both the Promalin-treated and control fruits were hyperallometric (i.e., faster than the increase in the center diameter). Again, the constant differential growth ratios were higher for Promalin-treated fruits than for the controls. The increases in the calyx region length over the same time period were nearly isoallometric for the Promalin-treated fruits but hypoallometric for the control fruits (Fig. 3F and 3H and Table 1).

The CM, DCM, and wax masses increased with time in the pedicel, center, and calyx regions (Fig. 4). The masses of CM (+3.3%) and DCM (+4.5%) of the Promalin-treated fruits were slightly but significantly higher than those of the control fruits. There were no significant differences in the wax mass of the Promalin-treated and the control fruits (Fig. 4). Additionally, there was no significant difference in the masses of the CM, DCM or wax of different regions of the fruits. Biaxial strain release on excision of the skin segments and isolation of CM (ɛexc+isol), on wax extraction from the CM (ɛextr) and the total strain release (ɛtot) increased during development but did not differ between regions on the fruit surface (Fig. 5). Small but significant differences in ɛexc+isol (1.4% vs. 1.9%), ɛextr (27.8% vs. 28.3%), and ɛtot (29.8% vs. 30.2%) between the Promalin-treated and control fruits were observed.

Fig. 4.
Fig. 4.

Time course of the change in the mass per unit area of the cuticular membrane (CM) (A–C), dewaxed CM (DCM) (D–F), and wax (G–I) in the pedicel (A, D, and G), center (B, E, and H), and calyx (C, F, and I) regions of control and Promalin-treated ‘Pinova’ apple fruits. Scale of x-axes = days after full bloom (DAFB).

Citation: HortScience 59, 7; 10.21273/HORTSCI17870-24

Fig. 5.
Fig. 5.

Time course of biaxial strain release of the cuticular membrane (CM) on excision of an exocarp segment (ES) and isolation of the CM (ɛexc+iso), on wax extraction from the CM (ɛextr), and the sum of the two strains (ɛtot) of untreated control and Promalin-treated ‘Pinova’ apple fruits in the pedicel (A, D, and G), center (B, E, and H), and calyx (C, F, and I) regions. Scale of x-axes = days after full bloom (DAFB).

Citation: HortScience 59, 7; 10.21273/HORTSCI17870-24

Ablation of the CM from the inner surface using CAPP reduced the mass of CM, DCM, and wax linearly with time as the duration of exposure increased (Fig. 6A and 6B). Additionally, the release of biaxial strain on wax extraction of the ablated (from the inner side) CM (ɛextr(CAPP)) increased with the CAPP exposure time for both the Promalin-treated and control fruits. For durations of CAPP longer than 10 min, the ɛextr(CAPP) in the calyx region was consistently higher for the control fruits than for the Promalin-treated fruits (Fig. 6C). When comparing ɛextr(CAPP) after 25 min of CAPP between different regions, the ɛextr(CAPP) values in the pedicel and the center regions were similar for Promalin-treated and control fruits (Table 2). For the calyx region, strain relaxation was somewhat larger for the control than for the Promalin-treated fruits. However, this difference was not significant.

Fig. 6.
Fig. 6.

Effect of the duration of cold atmospheric pressure plasma (CAPP) treatment of the morphological inner surfaces of cuticular membranes (CM) isolated from untreated control or Promalin-treated apple fruits on CM mass per unit area (A), the loss in CM mass per unit area (B), and the strain release following wax extraction (ɛextr(CAPP)) (C). The CMs were isolated from the calyx region of mature ‘Pinova’ apple fruits.

Citation: HortScience 59, 7; 10.21273/HORTSCI17870-24

Table 2.

Strain relaxation (ɛextr(CAPP)) of the cuticle of ‘Pinova’ apple fruits before (0 min) and after (25 min) ablation using cold atmospheric pressure plasma (CAPP). Strain relaxation was quantified in three different regions (pedicel, center, calyx) after wax extraction. Fruits were treated with Promalin, whereas untreated fruits served as controls.

Table 2.

There were essentially no microcracks in the cuticle of both Promalin-treated and control fruits, as indexed by infiltration with acridine orange (data not shown). The water vapor permeance of the skin was also similar in control and Promalin-treated fruits (4.7 ± 0.3 × 10−5 vs. 5.0 ± 0.2 × 10−5 m⋅s−1 for control vs. Promalin-treated fruits at 69 DAFB; 6.6 ± 0.6 × 10−5 vs. 6.9 ± 0.7 × 10−5 m⋅s−1 for control vs. Promalin-treated fruits at 147 DAFB).

Discussion

Our results demonstrate that Promalin stimulates apple fruit growth, mainly in the calyx and pedicel regions, with only small effects on cuticular deposition and cuticular strain relaxation. It has been previously reported that early-season Promalin applications increase the size and length of apple fruits by stimulating growth in the calyx region, primarily at the base of the sepals (Curry and Williams 1983). Images of the fruit cross-sections and the allometric growth analysis results presented in this work are consistent with those of Curry and Williams (1983). In addition, our data showed enhanced and increased lengths of the pedicel region. This finding has not been reported previously.

The CM and DCM masses per unit area in all regions of the surface of young fruits were only slightly higher in the Promalin-treated fruits than in the control fruits. Previous studies established that GA4 + 7 (a component of Promalin) increases the density of epidermal cells (cell number per unit area) (Curry 2012), but that it has no effect on cutin or wax deposition in ‘Golden Delicious’ apples (Knoche et al. 2011). The second active component in Promalin is 6-BA, which also stimulates cell division in much the same way as GA4 + 7 does. We are unaware of any reports of the effects of 6-BA on cuticle deposition in apples. From this information, it is reasonable to infer that our Promalin-treated apples will have more epidermal cells per unit of surface area (Curry 2012; Ginzberg et al. 2014). Our study also showed that Promalin causes a small but significant increase in mass per unit of surface area of the CM (+3.3%) and of the DCM (+4.5%). A possible explanation for this small effect on cuticle deposition may be more cuticular pegs above the anticlinal cell walls attributable to the likely larger number of epidermal cells per unit of fruit surface area. More epidermal cells per unit area would have a larger cumulative periclinal perimeter and, hence, a larger cumulative length of anticlinal cell walls. However, definitive evidence is lacking.

Similarly, relaxations of the excised CM and extracted CM and the sum of the two were marginally but significantly reduced in the Promalin-treated fruits compared with the control fruits. Interestingly, a comparison of the control fruits with the Promalin-treated fruits showed that there was a small and significant difference in the release of strain in the outer layers of the cutin matrix after CAPP ablation of the inner layers (ɛextr(CAPP)). It is known that apple deposits cutin on the morphological inner surface of the cuticle (Si et al. 2021). Thus, CAPP ablation of the inner cuticle surface removes the younger (more recently deposited) layers of the cuticle. Because strain relaxation increased as the duration of CAPP increased, the morphological outer layers of the CM must have been more strained than the inner layers. This finding is consistent with previous observations that demonstrated a radial gradient in the accumulated strain in apple cuticles. The (older) outer layers of the CM are more strained than the (younger) inner layers (Khanal et al. 2014; Si et al. 2021). On the inner surface, the more recent deposition of cuticular material prevents relaxation of the strain in the layers toward the outside; this effectively “fixes” that earlier strain (Khanal et al. 2013). The slightly reduced relaxation of cuticular strain in the Promalin-treated fruits (compared to that in the control fruits) after CAPP ablation of the inner cuticle layers is consistent with a slightly higher rate of deposition of cuticular material.

Conclusion

Promalin increased fruit size by stimulating growth in the pedicel and calyx regions. However, an increase in russeting was prevented by the deposition of the cuticle and fixation of strain, as stated in our second hypothesis. From a horticultural point of view, Promalin-treated fruits and untreated control fruits are similar in terms of strain relaxation, microcracking, and susceptibility to russeting.

References Cited

  • Curry EA. 2012. Increase in epidermal planar cell density accompanies decreased russeting of ‘Golden Delicious’ apples treated with gibberellin A4 + 7. HortScience. 47(2):232237. https://doi.org/10.21273/HORTSCI.47.2.232.

    • Search Google Scholar
    • Export Citation
  • Curry EA, Williams MW. 1983. Promalin or GA3 increase pedicel and fruit length and leaf size of ‘Delicious’ apples treated with paclobutrazol. HortScience. 18(2):214215. https://doi.org/10.21273/HORTSCI.18.2.214.

    • Search Google Scholar
    • Export Citation
  • Eccher T, Boffelli G. 1981. Effects of dose and time of application of GA4 + 7 on russeting, fruit Set and shape of ‘Golden Delicious’ apples. Scientia Hortic. 14:307314. https://doi.org/10.1016/0304-4238(81)90043-1.

    • Search Google Scholar
    • Export Citation
  • Geyer U, Schönherr J. 1988. In vitro test for effects of surfactants and formulations on permeability of plant cuticles, p 22–33. In: Cross B, Scher HB (eds). Pesticide formulations: Innovations and developments. American Chemical Society, Washington, DC, USA.

  • Ginzberg I, Stern RA. 2016. Strengthening fruit-skin resistance to growth strain by application of plant growth regulators. Scientia Hortic. 198:150153. https://doi.org/10.1016/j.scienta.2015.11.016.

    • Search Google Scholar
    • Export Citation
  • Ginzberg I, Fogelman E, Rosenthal L, Stern RA. 2014. Maintenance of high epidermal cell density and reduced calyx-end cracking in developing ‘Pink Lady’ apples treated with a combination of cytokinin 6-benzyladenine and gibberellins A4+A7. Scientia Hortic. 165:324330. https://doi.org/10.1016/j.scienta.2013.11.020.

    • Search Google Scholar
    • Export Citation
  • Huxley JS. 1924. Constant differential growth-ratios and their significance. Nature. 117:895896. https://doi.org/10.1038/114895a0.

  • Khanal BP, Bhattarai A, Aryal D, Knoche M. 2023. Neck shrivel in European plum is caused by cuticular microcracks, resulting from rapid lateral expansion of the neck late in development. Planta. 258:62. https://doi.org/10.1007/s00425-023-04218-9.

    • Search Google Scholar
    • Export Citation
  • Khanal BP, Grimm E, Finger S, Blume A, Knoche M. 2013. Intracuticular wax fixes and restricts strain in leaf and fruit cuticles. New Phytol. 200:134143. https://doi.org/10.1111/nph.12355.

    • Search Google Scholar
    • Export Citation
  • Khanal BP, Knoche M, Bußler S, Schlüter O. 2014. Evidence for a radial strain gradient in apple fruit cuticles. Planta. 240:891897. https://doi.org/10.1007/s00425-014-2132-0.

    • Search Google Scholar
    • Export Citation
  • Knoche M, Khanal BP, Stopar M. 2011. Russeting and microcracking of ‘Golden Delicious’ apple fruit concomitantly decline due to Gibberellin A4 + 7 application. J Am Soc Hortic Sci. 136(3):159164. https://doi.org/10.21273/JASHS.136.3.159.

    • Search Google Scholar
    • Export Citation
  • Knoche M, Peschel S, Hinz M, Bukovac MJ. 2000. Studies on water transport through the sweet cherry fruit surface: Characterizing conductance of the cuticular membrane using pericarp segments. Planta. 212:127135. https://doi.org/10.1007/s004250000404.

    • Search Google Scholar
    • Export Citation
  • Lai X, Khanal BP, Knoche M. 2016. Mismatch between cuticle deposition and area expansion in fruit skins allows potentially catastrophic buildup of elastic strain. Planta. 244:11451156. https://doi.org/10.1007/s00425-016-2572-9.

    • Search Google Scholar
    • Export Citation
  • Leite GB, Petri JL, Basso C. 2006. Promalin effect on ‘Imperial Gala’ and ‘Fuji’ apple trees fructification. Acta Hortic. 727:269278. https://doi.org/10.17660/ActaHortic.2006.727.31.

    • Search Google Scholar
    • Export Citation
  • Nobel PS. 1999. Physicochemical & Environmental Plant Physiology (4th ed). Academic Press, San Diego, CA, USA.

  • Scharwies JD, Grimm E, Knoche M. 2014. Russeting and relative growth rate are positively related in ‘Conference’ and ‘Condo’ pear. HortScience. 49(6):746749. https://doi.org/10.21273/HORTSCI.49.6.746.

    • Search Google Scholar
    • Export Citation
  • Si Y, Khanal BP, Schlüter OK, Knoche M. 2021. Direct evidence for a radial gradient in age of the apple fruit cuticle. Front Plant Sci. 12:730837. https://doi.org/10.3389/fpls.2021.730837.

    • Search Google Scholar
    • Export Citation
  • Skene DS. 1966. The distribution of growth and cell division in the fruit of Cox’s Orange Pippin. Ann Bot. 30:493512. https://doi.org/10.1093/oxfordjournals.aob.a084092.

    • Search Google Scholar
    • Export Citation
  • Veinbrants N, Miller P. 1981. Promalin improves the shape of Delicious apples in Victoria. Aust J Exp Agric. 21:623630. https://doi.org/10.1071/EA9810623.

    • Search Google Scholar
    • Export Citation
  • Fig. 1.

    Developmental time course of the change in the surface area (A), length (B), and diameter (C) of the control and Promalin-treated ‘Pinova’ apple fruits. Inset in (A) upper left: Time course of change in the surface area growth rate. Inset in (A) lower right: Time course of change in the surface area relative growth rate. Inset in (B): Time course of change in the length-to-diameter ratio. Scale of x-axes in days after full bloom (DAFB). Representative images of control (D) and Promalin-treated ‘Pinova’ apple fruits (E) at maturity. Data points represent the mean ± SE. Bar = 2 cm.

  • Fig. 2.

    Longitudinal cross-sections of untreated control (A–F) and Promalin-treated (G–L) ‘Pinova’ apple fruits. Fruits were harvested at various stages of development. The scale bar in (A) is applicable to (A, B, D, E, H, J, and K). The scale bar in (C) applies to (C and I). The scale bar in (F) applies to (F and L). DAFB = days after full bloom.

  • Fig. 3.

    (A) Sketch of the longitudinal cross-section through an apple fruit illustrating the nomenclature used to describe the different dimensions of the fruit in the calyx, center, and pedicel regions. (B–D) Relationship between the log-transformed diameters in the pedicel region (B), core (C), and calyx region (D) and log-transformed center diameter of the fruit. (E–H) Relationship between the log-transformed lengths of the whole fruit (E), pedicel region length (F), core length (G), and calyx region length (H) and log-transformed center diameter of the fruit. The slopes (constant differential growth ratios) and intercepts of these relationships are shown in Table 1.

  • Fig. 4.

    Time course of the change in the mass per unit area of the cuticular membrane (CM) (A–C), dewaxed CM (DCM) (D–F), and wax (G–I) in the pedicel (A, D, and G), center (B, E, and H), and calyx (C, F, and I) regions of control and Promalin-treated ‘Pinova’ apple fruits. Scale of x-axes = days after full bloom (DAFB).

  • Fig. 5.

    Time course of biaxial strain release of the cuticular membrane (CM) on excision of an exocarp segment (ES) and isolation of the CM (ɛexc+iso), on wax extraction from the CM (ɛextr), and the sum of the two strains (ɛtot) of untreated control and Promalin-treated ‘Pinova’ apple fruits in the pedicel (A, D, and G), center (B, E, and H), and calyx (C, F, and I) regions. Scale of x-axes = days after full bloom (DAFB).

  • Fig. 6.

    Effect of the duration of cold atmospheric pressure plasma (CAPP) treatment of the morphological inner surfaces of cuticular membranes (CM) isolated from untreated control or Promalin-treated apple fruits on CM mass per unit area (A), the loss in CM mass per unit area (B), and the strain release following wax extraction (ɛextr(CAPP)) (C). The CMs were isolated from the calyx region of mature ‘Pinova’ apple fruits.

  • Curry EA. 2012. Increase in epidermal planar cell density accompanies decreased russeting of ‘Golden Delicious’ apples treated with gibberellin A4 + 7. HortScience. 47(2):232237. https://doi.org/10.21273/HORTSCI.47.2.232.

    • Search Google Scholar
    • Export Citation
  • Curry EA, Williams MW. 1983. Promalin or GA3 increase pedicel and fruit length and leaf size of ‘Delicious’ apples treated with paclobutrazol. HortScience. 18(2):214215. https://doi.org/10.21273/HORTSCI.18.2.214.

    • Search Google Scholar
    • Export Citation
  • Eccher T, Boffelli G. 1981. Effects of dose and time of application of GA4 + 7 on russeting, fruit Set and shape of ‘Golden Delicious’ apples. Scientia Hortic. 14:307314. https://doi.org/10.1016/0304-4238(81)90043-1.

    • Search Google Scholar
    • Export Citation
  • Geyer U, Schönherr J. 1988. In vitro test for effects of surfactants and formulations on permeability of plant cuticles, p 22–33. In: Cross B, Scher HB (eds). Pesticide formulations: Innovations and developments. American Chemical Society, Washington, DC, USA.

  • Ginzberg I, Stern RA. 2016. Strengthening fruit-skin resistance to growth strain by application of plant growth regulators. Scientia Hortic. 198:150153. https://doi.org/10.1016/j.scienta.2015.11.016.

    • Search Google Scholar
    • Export Citation
  • Ginzberg I, Fogelman E, Rosenthal L, Stern RA. 2014. Maintenance of high epidermal cell density and reduced calyx-end cracking in developing ‘Pink Lady’ apples treated with a combination of cytokinin 6-benzyladenine and gibberellins A4+A7. Scientia Hortic. 165:324330. https://doi.org/10.1016/j.scienta.2013.11.020.

    • Search Google Scholar
    • Export Citation
  • Huxley JS. 1924. Constant differential growth-ratios and their significance. Nature. 117:895896. https://doi.org/10.1038/114895a0.

  • Khanal BP, Bhattarai A, Aryal D, Knoche M. 2023. Neck shrivel in European plum is caused by cuticular microcracks, resulting from rapid lateral expansion of the neck late in development. Planta. 258:62. https://doi.org/10.1007/s00425-023-04218-9.

    • Search Google Scholar
    • Export Citation
  • Khanal BP, Grimm E, Finger S, Blume A, Knoche M. 2013. Intracuticular wax fixes and restricts strain in leaf and fruit cuticles. New Phytol. 200:134143. https://doi.org/10.1111/nph.12355.

    • Search Google Scholar
    • Export Citation
  • Khanal BP, Knoche M, Bußler S, Schlüter O. 2014. Evidence for a radial strain gradient in apple fruit cuticles. Planta. 240:891897. https://doi.org/10.1007/s00425-014-2132-0.

    • Search Google Scholar
    • Export Citation
  • Knoche M, Khanal BP, Stopar M. 2011. Russeting and microcracking of ‘Golden Delicious’ apple fruit concomitantly decline due to Gibberellin A4 + 7 application. J Am Soc Hortic Sci. 136(3):159164. https://doi.org/10.21273/JASHS.136.3.159.

    • Search Google Scholar
    • Export Citation
  • Knoche M, Peschel S, Hinz M, Bukovac MJ. 2000. Studies on water transport through the sweet cherry fruit surface: Characterizing conductance of the cuticular membrane using pericarp segments. Planta. 212:127135. https://doi.org/10.1007/s004250000404.

    • Search Google Scholar
    • Export Citation
  • Lai X, Khanal BP, Knoche M. 2016. Mismatch between cuticle deposition and area expansion in fruit skins allows potentially catastrophic buildup of elastic strain. Planta. 244:11451156. https://doi.org/10.1007/s00425-016-2572-9.

    • Search Google Scholar
    • Export Citation
  • Leite GB, Petri JL, Basso C. 2006. Promalin effect on ‘Imperial Gala’ and ‘Fuji’ apple trees fructification. Acta Hortic. 727:269278. https://doi.org/10.17660/ActaHortic.2006.727.31.

    • Search Google Scholar
    • Export Citation
  • Nobel PS. 1999. Physicochemical & Environmental Plant Physiology (4th ed). Academic Press, San Diego, CA, USA.

  • Scharwies JD, Grimm E, Knoche M. 2014. Russeting and relative growth rate are positively related in ‘Conference’ and ‘Condo’ pear. HortScience. 49(6):746749. https://doi.org/10.21273/HORTSCI.49.6.746.

    • Search Google Scholar
    • Export Citation
  • Si Y, Khanal BP, Schlüter OK, Knoche M. 2021. Direct evidence for a radial gradient in age of the apple fruit cuticle. Front Plant Sci. 12:730837. https://doi.org/10.3389/fpls.2021.730837.

    • Search Google Scholar
    • Export Citation
  • Skene DS. 1966. The distribution of growth and cell division in the fruit of Cox’s Orange Pippin. Ann Bot. 30:493512. https://doi.org/10.1093/oxfordjournals.aob.a084092.

    • Search Google Scholar
    • Export Citation
  • Veinbrants N, Miller P. 1981. Promalin improves the shape of Delicious apples in Victoria. Aust J Exp Agric. 21:623630. https://doi.org/10.1071/EA9810623.

    • Search Google Scholar
    • Export Citation
Bishnu P. Khanal Institute of Horticultural Production Systems, Fruit Science Section, Leibniz University Hannover, Herrenhäuser Straβe 2, 30419 Hannover, Germany

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Prabin Dahal Institute of Horticultural Production Systems, Fruit Science Section, Leibniz University Hannover, Herrenhäuser Straβe 2, 30419 Hannover, Germany

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Yiru Si Institute of Horticultural Production Systems, Fruit Science Section, Leibniz University Hannover, Herrenhäuser Straβe 2, 30419 Hannover, Germany

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Moritz Knoche Institute of Horticultural Production Systems, Fruit Science Section, Leibniz University Hannover, Herrenhäuser Straβe 2, 30419 Hannover, Germany

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Oliver K. Schlüter Department of System Process Engineering, Leibniz Institute for Agricultural Engineering and Bioeconomy (ATB), Max-Eyth-Allee 100, 14469 Potsdam, Germany

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Contributor Notes

The publication of this article was funded in part by the Open Access Fund of Leibniz Universität Hannover. We thank Dr. Alexander Lang and Dr. Andreas Winkler for helpful discussions of and useful comments regarding an earlier version of this manuscript. We also thank Dr. Michael Schröder (Sumitomo Chemical, EMEA region), Dr. Peter D. Petracek, and Dr. Steven McArtney (Valent BioSciences Corporation) for helpful suggestions and the gift of Promalin®.

B.P.K. is the corresponding author. E-mail: khanal@obst.uni-hannover.de.

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  • Fig. 1.

    Developmental time course of the change in the surface area (A), length (B), and diameter (C) of the control and Promalin-treated ‘Pinova’ apple fruits. Inset in (A) upper left: Time course of change in the surface area growth rate. Inset in (A) lower right: Time course of change in the surface area relative growth rate. Inset in (B): Time course of change in the length-to-diameter ratio. Scale of x-axes in days after full bloom (DAFB). Representative images of control (D) and Promalin-treated ‘Pinova’ apple fruits (E) at maturity. Data points represent the mean ± SE. Bar = 2 cm.

  • Fig. 2.

    Longitudinal cross-sections of untreated control (A–F) and Promalin-treated (G–L) ‘Pinova’ apple fruits. Fruits were harvested at various stages of development. The scale bar in (A) is applicable to (A, B, D, E, H, J, and K). The scale bar in (C) applies to (C and I). The scale bar in (F) applies to (F and L). DAFB = days after full bloom.

  • Fig. 3.

    (A) Sketch of the longitudinal cross-section through an apple fruit illustrating the nomenclature used to describe the different dimensions of the fruit in the calyx, center, and pedicel regions. (B–D) Relationship between the log-transformed diameters in the pedicel region (B), core (C), and calyx region (D) and log-transformed center diameter of the fruit. (E–H) Relationship between the log-transformed lengths of the whole fruit (E), pedicel region length (F), core length (G), and calyx region length (H) and log-transformed center diameter of the fruit. The slopes (constant differential growth ratios) and intercepts of these relationships are shown in Table 1.

  • Fig. 4.

    Time course of the change in the mass per unit area of the cuticular membrane (CM) (A–C), dewaxed CM (DCM) (D–F), and wax (G–I) in the pedicel (A, D, and G), center (B, E, and H), and calyx (C, F, and I) regions of control and Promalin-treated ‘Pinova’ apple fruits. Scale of x-axes = days after full bloom (DAFB).

  • Fig. 5.

    Time course of biaxial strain release of the cuticular membrane (CM) on excision of an exocarp segment (ES) and isolation of the CM (ɛexc+iso), on wax extraction from the CM (ɛextr), and the sum of the two strains (ɛtot) of untreated control and Promalin-treated ‘Pinova’ apple fruits in the pedicel (A, D, and G), center (B, E, and H), and calyx (C, F, and I) regions. Scale of x-axes = days after full bloom (DAFB).

  • Fig. 6.

    Effect of the duration of cold atmospheric pressure plasma (CAPP) treatment of the morphological inner surfaces of cuticular membranes (CM) isolated from untreated control or Promalin-treated apple fruits on CM mass per unit area (A), the loss in CM mass per unit area (B), and the strain release following wax extraction (ɛextr(CAPP)) (C). The CMs were isolated from the calyx region of mature ‘Pinova’ apple fruits.

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