Plant material.

Sweet cherry fruit of the cultivars Sam, Regina, Adriana, Dönissens Gelbe, Frühe Rote Mecklenburger, Hedelfinger, Kordia, Burlat, Merchant, Staccato, and Sweetheart were picked randomly from a minimum of three trees grafted on ‘Gisela 5’ rootstocks (Prunus cerasus L. × P. canescens Bois) grown under a rain shelter in the field or in an experimental greenhouse at the horticultural experiment station at Ruthe (lat. 52°14′ N, long. 9°49′ E) in the 2012, 2013, and 2014 seasons. Fruit were harvested early in the mornings and selected for absence of visible defects and for uniformity of maturity based on skin color and processed within 2 h.

General experimental procedure.

Fruit with and without their pedicels and/or detached excised pedicels were incubated at 22 °C in sealed polyethylene (PE) boxes at a defined water vapor concentration. Unless specified otherwise, pedicel length of fruit with pedicels was maintained constant within any one experiment by cutting the proximal portion using a razor blade. Fruit without pedicels were prepared by cutting their pedicels proximal of the receptacle and sealing the stem/fruit juncture and the receptacle including the pedicel end using an epoxy glue (UHU plus schnellfest; UHU, Bühl/Baden, Germany). Water loss was recorded by repeated weighings of individual fruit and/or pedicels. Rates of transpiration (F) were calculated for each fruit or (detached) pedicel as the slope of a linear regression through a plot of cumulative mass loss against time. Pedicel transpiration was quantified by difference. This involved subtracting the average rate of transpiration of fruit without their pedicels from that of fruit with their pedicels. A preliminary experiment established that this method was straightforward and also yielded essentially the same values for pedicel transpiration rate as from less straightforward, but more direct, gravimetric determinations (see immediately below) of the amounts of water transpired by attached pedicels [3.6 ± 0.2 vs. 4.2 ± 0.3 mg·h⁻¹ (n = 10, P < 0.139)]. The more direct determination of transpiration from fruit and also from their attached pedicels was accomplished in this preliminary experiment by inserting a still-attached pedicel through an orifice (3 mm diameter) in the lid of a falcon tube (29 mm diameter, 115 mm long) filled with dry silica gel and the whole assembly sealed in a PE box that also contained dry silica gel. In this way, the silica in the falcon tube served as a receiver for water transpired from the pedicel surface and that in the PE box as a receiver for water transpired from the fruit surface. Also, in contrast to salts, the silica in the falcon tube allowed the pedicel to be removed, fruit with pedicel weighed, and the pedicel to be re-inserted without any contamination of the pedicel surface. At selected intervals, fruit were removed from the PE box and from the falcon tube and both, fruit and the falcon tube (without the fruit), weighed. Rates of transpiration were calculated as described previously. From the rates of transpiration of fruit (F_fruit) and pedicels (F_pedicel), the permeances of the fruit (p_fruit) and pedicel surfaces (p_pedicel) were calculated. The value A_fruit was calculated from fruit mass assuming a mean density of 1 kg·dm⁻³ and a spherical shape. Meanwhile, the value A_pedicel was calculated from pedicel length and mean diameter [measured at the midpoint using digital calipers (CD-30PK; Mitutoyo, Kawasaki/Kanagawa, Japan)] and assuming a cylindrical shape. The (grams per cubic meter) represents the difference in water vapor concentration between the atmosphere within the fruit, or the pedicel, and that inside the PE box. The water vapor concentration inside the fruit and inside the pedicel is essentially saturated [23.07 g·m⁻³ at 22 °C (Nobel, 1999)]. With few exceptions where fruit and/or pedicels were incubated above silica or water, the PE box contained saturated salt slurries to establish defined humidities (Wexler, 1995). For example, saturated NaCl, used in most experiments, generates a water vapor concentration of 14.6 g·m⁻³ at 22 °C. The water vapor concentration gradient driving transpiration of fruit and/or pedicel inside the PE box amounts to 8.47 g·m⁻³.

Experiments.

The time course of water loss from fruit and pedicels was established over a 96-h period using mature ‘Staccato’ sweet cherry incubated above NaCl. Pedicels were carefully detached at the spur and remained at their full length. The number of single fruit replicates was 12.

To identify the source of the water transpired by the pedicel, transpiration was monitored for excised pedicels and pedicels that remained attached to their fruit (all above NaCl). Pedicel length was standardized to 40 mm by cutting using a razor blade. The cultivar was Frühe Rote Mecklenburger. The number of individual fruit replicates was 10.

The vascular system (xylem or phloem) that supplies the pedicel with water was identified by studying the effect of steam girdling and the effect of pedicel orientation in two separate experiments. In the first, the effect of steam girdling was quantified by monitoring transpiration of girdled and non-girdled pedicels (30 mm length) of ‘Merchant’ sweet cherry above NaCl. The pedicels were girdled above the receptacle using a modified steam generator [width of girdle 5 mm (SC952; Alfred Kärcher, Winnenden, Germany)]. There was no contact between the steam and the fruit surface. Transpiration was quantified by repeated weighing of fruit with and without the pedicel as described previously. Fruit with non-girdled pedicels served as controls. The number of single fruit replicates was 10. In the second experiment the effect of pedicel orientation was studied using excised pedicels of ‘Frühe Rote Mecklenburger’. Pedicels were inserted through an orifice (2.2 mm diameter) in the lid of a 2-mL Eppendorf tube (CK 06.1; Carl Roth, Karlsruhe, Germany) such that either the basal or the apical end was immersed in water. The transpiring portion of the pedicel extended 25 mm beyond the seal. We refer to these orientations as the normal (basal end immersed in water in the Eppendorf tube) or reverse orientation (apical end immersed). To avoid cavitation, the pedicels were recut under water before being immersed in the Eppendorf tube. The tubes with pedicels inserted were incubated above NaCl. The number of replicates was 10 where one replicate represented one pedicel.

The rate-limiting step in pedicel transpiration was investigated by modifying the pedicel surface using the following treatments. The cuticle was abraded from the surface of ‘Regina’ sweet cherry pedicels (40 mm length by razor blade) using abrasive paper (grit 600; Emil Lux, Wermelskirchen, Germany). Waxes were extracted by dipping excised pedicels once or five times for 10 s each in CHCl₃/MeOH (1:1 v/v). Pedicel ends were sealed with fast-curing epoxy glue. Transpiration was quantified above NaCl by repeated weighing of the pedicels over a 1-h period. The number of single pedicel replicates was 10.

Differences in pedicel transpiration among cultivars were studied in Adriana, Burlat, Dönissens Gelbe, Frühe Rote Mecklenburger, Hedelfinger, Kordia, Regina, Sam, and Sweetheart. Pedicels were carefully detached at the spur and remained at their full length. Transpiration of fruit and pedicel incubated above NaCl was determined using 10 replicates. To relate pedicel transpiration to morphological characteristics, pedicel length, diameter, and stomatal densities were determined. Briefly, pedicel sections were photographed under a dissecting microscope (MZ6; Leica Mikrosysteme, Bensheim, Germany) and length and width quantified by image analysis (Cell^P; Olympus Europa, Hamburg, Germany). To establish the distribution of stomata along the pedicel, ‘Sam’ pedicels were cut into five sections each ≈8 mm in length, which were then cut along their long axes into two halves and incubated in 50 mm citric acid buffer solution (pH 4.0) containing 90 mL·L⁻¹ pectinase (Panzym Super E flüssig; Novozymes, Bagsvaerd, Denmark), 5 mL·L⁻¹ cellulase (Cellubrix L; Novozymes), and NaN₃ at 30 mm (Orgell, 1955; Yamada et al., 1964). After ≈14 d, the CMs had separated from adhering cell layers. The CMs were then spread on microscope slides, transferred to the stage of a fluorescence microscope (BX-60; Olympus Europa), and observed at × 100. The number of stomata was determined in a representative area (range, 5 to 17 mm²) of both halves of each segment. Stomatal density was calculated by dividing the number of stomata by the area counted. Preliminary experiments established that there was no significant strain relaxation of the CM strips after isolation. For the cultivar comparison, stomatal densities were determined for the midportion of the pedicel. Total stomata number per pedicel was calculated by multiplying the density of stomata in the midportion by the surface area of the whole pedicel. Pedicel dimensions and stomatal densities were determined using five replicates.

The role of stomata in pedicel transpiration was addressed using two different approaches. In the first approach, ‘Staccato’ fruit with and without pedicel (length 40 mm) were incubated in PE boxes closed with transparent Saran wrapping (Frischhaltefolie; REWE Handelsgruppe, Cologne, Germany). The PE boxes were continuously flushed with CO₂, N₂, or ambient air after passing through dry silica. These atmospheres are expected to reduce, increase, or not affect (respectively) the extent of stomatal opening (F. Lenz, personal communication). Pedicel transpiration was quantified over a 5-h interval (n = 5). For the second approach, ‘Frühe Rote Mecklenburger’ pedicels (40 mm) with fruit still attached were incubated for 1 h into 0.1 mm abscisic acid (ABA) prepared in 10 mm citric acid buffer at pH 3.6. Buffer solution without ABA served as a control. Transpiration of fruit with and without pedicels was monitored gravimetrically over a 6- h period as described previously. The number of single fruit replicates was 10.

The effect of temperature on ‘Sam’ fruit and pedicel transpiration was investigated after a 24-h incubation period at 2, 12, 22, and 35 °C above dry silica gel. The number of individual fruit replicates was 15.

To test the effect of RH, ‘Sam’ fruit with and without pedicels (30 mm length) were incubated above dry silica gel [0% RH (Geyer and Schönherr, 1988)], saturated slurries of CaCl₂ (28% RH), NaCl (75% RH), KNO₃ [92% RH (Wexler, 1995)], or above water (100% RH). The number of individual fruit replicates was 10.

Data analysis.

Data in tables and figures are presented as means ±se. The following equation was used to calculate ses of pedicel transpiration (z) obtained by the difference from the mean transpiration of fruit with pedicels (x₁) minus that without pedicels (x₂) (Sachs, 1992):

View Expanded

Where error bars are not visible, they are smaller than the data symbols.

The fruit sustains pedicel transpiration through the xylem.

A fruit serves as a water supply to sustain the transpiration of an attached pedicel. This conclusion is supported by several arguments. First, pedicels that remained attached to the fruit had an approximate constant rate of water loss up to ≈4 h, when rates began to decrease somewhat (Figs. 1B and 2). Indeed, the cumulative transpiration of pedicels soon exceeded their own total water contents, indicating that the water they transpired must have been obtained from the fruit (Fig. 2). Second, initial rates of transpiration were similar for excised and for attached pedicels (Fig. 2 inset). From this observation we infer that initial rates of transpiration are governed by properties of the pedicel surface rather than by any restriction of water supply imposed by the vascular connection to the fruit. The decline in the transpiration rates of excised pedicels after some hours is explained by their limited fresh weight and, consequently, the small amount of available water they contain.

Water transfer from fruit to a transpiring pedicel occurs mainly through the xylem. This conclusion is based on the lack of any effect of steam girdling on pedicel transpiration and also on the lack of an effect of pedicel orientation. Steam girdling interrupts phloem transport by killing its cells. In contrast, xylem conductance is unaffected by steam girdling and is independent of the direction of water flow (Lang and Thorpe, 1989). It may be argued that the formation of an abscission layer at the fruit/pedicel juncture could affect postharvest pedicel transpiration by affecting the conductance of the vascular system. However, studies with a number of cultivars have shown that formation of an abscission layer (as indexed by a declining pedicel–fruit retention force) is not related to any change in vascular connectivity between a fruit and its pedicel (Smith and Whiting, 2011). Also, in earlier anatomical studies there is no evidence that an abscission layer might interrupt the vascular bundles of mature ‘Windsor’ sweet cherries (Wittenbach and Bukovac, 1972). This would be a prerequisite for any effect of an abscission layer on pedicel transpiration. Based on these arguments, we do not expect abscission layer formation to be a significant factor in regulating pedicel transpiration.

Pedicel transpiration obeyed fick’s law.

Water movement through the cuticular membrane of the pedicel surface was the rate-limiting step in transpiration. The effects of various factors on transpiration rate were accounted for by their effects either on permeance and/or on the driving force.

On average, the permeance of the pedicel surface exceeded that of the fruit 4-fold. It may be argued that because the pedicel surface is stomatous, this p is a composite (in parallel) property comprising both stomatal and cuticular conductance components. Furthermore, the stomatal p component is expected to be a function of stomatal density and/or stomatal pore area. Stomatal densities on pedicel surfaces were on average 40-fold higher than on fruit surfaces (Table 2; Peschel et al., 2003; Peschel and Knoche, 2012). However, there is evidence here that stomata did not play a major role in determining pedicel transpiration. First, there was no significant relationship between total stomatal number and the rate of transpiration or between stomatal density and the p of the pedicel surface (Fig. 3C and D). Second, none of the treatments known to induce stomatal closure or opening (such as dipping in ABA or incubation in CO₂- or N₂-atmospheres) had any effect on transpiration (Table 3). If stomata were functional, we would expect ABA or CO₂ atmosphere to decrease and N₂ to increase transpiration as compared with the non-treated control. Third, we were unable to detect any changes in stomatal aperture in the course of dehydration or in response to ABA treatments (T. Athoo, unpublished data). Fourth, the water potential of sweet cherry fruit is very negative as a result of the high concentration of carbohydrates and the absence of significant turgor (Knoche et al., 2014). This negative water potential would impose significant water stress on the fruit pedicel causing stomata to close (assuming they were functional). Stomata on the surface of mature fruit have been shown to be non-functional (Peschel et al., 2003). Finally, under best-practice postharvest conditions, packaged fruit is held in the dark, often in a high CO₂ atmosphere, and both factors are likely to close off any still-functional stomata.

The lack of a significant contribution of stomatal transpiration to total pedicel transpiration and the apparent absence of significant relationships between rates of transpiration and pedicel surface area in the between-cultivar comparison imply the permeability of the cuticle between stomata must differ significantly between cultivars. Such differences could be the result of differing amounts of wax or differences in the composition and crystallinity of the wax. It is clear that the wax and, within the wax, the fraction extracted by the first chloroform/methanol dip contribute the major resistances to transpiration. It may be speculated that the first dip primarily removed epicuticular wax, which is located on the cuticle surface and, hence, more readily accessible to the solvents. However, 1) solubility differs between wax constituents; and 2) solvents will also penetrate into the cuticle and both factors will limit spatial selectivity of the extraction procedure. Unfortunately, as a result of the fragility of the pedicel cuticle, conclusive direct evidence is difficult to obtain.

The effect of temperature and RH on transpiration is also accounted for by Fick’s law. Both factors alter the driving force for transpiration but neither had a significant effect on the permeance of the pedicel surface. Interestingly, when fruit with pedicels were held for several days, slight shriveling of the pedicel was observed even in a water-saturated atmosphere. According to Linke et al. (2010), pedicel shriveling is a linear function of the pedicel’s water content. However, the shriveling in the long-term incubation cannot be accounted for by transpiration, because fruit actually gained mass slightly under these conditions (T. Athoo, unpublished data). Thus, additional factors must have been involved. The mechanistic basis of pedicel dehydration under a water-saturated atmosphere is currently under investigation.