Plant material.

Fruits of uniform size and color were harvested at commercial maturity from greenhouse-grown sweet cherries (‘Kordia’, ‘Korvic’, ‘Sam’, ‘Samba’, ‘Sweetheart’, ‘Staccato’) and sour cherries (‘Ungarische Traubige’) both grafted on ‘Gisela 5’ rootstocks (P. cerasus × Prunus canescens Bois) or from field-grown european plum (‘Doppelte Hauspflaume’ grafted on Prunus insitia L. ‘St. Julien A’), gooseberry (‘Invicta’), red currant (‘Rovada’), black currant (‘Titania’), and blueberry (‘Elliott’) at the Horticultural Research Station in Sarstedt (lat. 52°14′ N, long. 9°49′ E) or the Herrenhausen campus garden of the Leibniz University Hannover (lat. 52°23′ N, long. 9°42′ E). Grapes were obtained from a commercial vineyard at Gleidingen [‘Fanny’ (lat. 52°16′ N, long. 9°42′ E)] or an experimental vineyard at Neustadt/Weinstraße [‘Riesling’ (lat. 49°22′ N, long. 8°11′ E)]. Tomato (‘Micro-Tom’) was cultivated in a greenhouse on the Herrenhausen campus garden. Fruit were processed on the day of sampling. The only exceptions were the ‘Riesling’ grapes that were processed within 34 h of sampling.

Quantifying and using pressure probes.

The pressure probe is a device for investigating plant water relations. For detailed descriptions of the various techniques involved and modes of operation, see Steudle (1993). The pressure probes used in our study were the cell pressure probe and the root pressure probe as described by Steudle (1993). The latter probe may also be used to quantify the in detached fruit (e.g., Lang and Düring, 1990) and is therefore referred to here as the fruit pressure probe. The functional principles of the CPP and FPP are identical. Both instruments comprise a drawn glass capillary (tip diameter 30 to 60 μm for the CPP and 150 to 250 μm for the FPP) filled with silicone oil (AS 4; Wacker Chemie, München, Germany) free of air bubbles and connected to a pressure sensor (26PCGFA6D; Honeywell Sensing and Control, Golden Valley, MN). When inserting the capillary of the CPP into a cell or that of the FPP into a fruit and establishing hydraulic continuity with the cell’s symplast (CPP), or the fruit flesh (FPP), the pressure inside the probe equilibrates with that in the cell (CPP), or fruit (FPP). In CPP experiments, successful insertions of the capillary are determined under a horizontal-axis microscope when a meniscus becomes visible in the capillary between the aqueous cell contents (forced into the capillary by cell pressure) and the silicone oil. This insertion is not associated with a marked change in pressure (Fig. 1). To make a measurement, the meniscus is pushed back usually after 10 to 15 s to its presumed initial position at the tip of the capillary by careful displacement of the oil. When the meniscus is in this position, the initial volume of the punctured cell is approximately restored. The push back is associated with an increase in pressure up to a peak pressure. Thereafter, the pressure decreases slightly approaching an asymptote at a slightly lower level (Fig. 1). The peak pressure recorded in the oil is deemed to represent the value . Peak pressure () and the pressure of the asymptote () are very closely and linearly related [ (kilopascals) = 0.79(± 0.007) × (kilopascals); r² = 0.99, P = 0.0001, n = 82]. Preliminary experiments established that there is no significant difference (P = 0.55) between the peak pressure (Stage II ‘Regina’: 234 ± 40 kPa) and the mean pressure (Stage II ‘Regina’: 273 ± 30 kPa) recorded when the meniscus is held just above the fruit surface (E. Grimm, unpublished data). For this experiment, Stage II fruit was selected, because at this developmental stage, the is markedly higher than at the mature stage (E. Grimm, unpublished data).

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Typical time course of change in pressure during a measurement of cell turgor of a single cell in the outer mesocarp of a mature ‘Samba’ sweet cherry fruit using the cell pressure probe (CPP). The glass capillary is inserted into a cell at 0 min (“Insert”) under a horizontal axis microscope. The juice/silicone oil meniscus is pushed back (“Push back”) to its original position at the tip of the capillary at 0.15 min (equivalent to 10 s). This push back restores the initial cell volume and is associated with an increase in pressure. The peak pressure recorded in the oil after the push back represents the . Thereafter, the pressure typically decreases slightly and then remains constant. At ≈1.5 min, the CPP is pressurized again (“Pressurize”) to control for plugging of the capillary by injecting silicone oil into the cell. After an instantaneous transient increase, the pressure re-equilibrates within ≈ 1 min and then drops to the ambient pressure (initial before the test) as the capillary is withdrawn from the cell at ≈2.5 min (“Withdraw”).

Citation: Journal of the American Society for Horticultural Science 139, 1; 10.21273/JASHS.139.1.3

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When using the FPP, insertion of the capillary is associated with an increase in the pressure signal (Fig. 2A). Unlike CPP measurements, a meniscus is not usually detectable. In this case an equilibrium pressure is recorded as the with no further manipulation. Given the tiny displacement volume of the probe apparatus relative to that of the whole fruit, this seems reasonable (additional care is clearly more important for the cell probe where the relative volumes are more similar). In our FPP measurements on tomato, a meniscus was usually visible in the capillary so, in this case, we were able to force this back to the tip before reading the equilibrium pressure.

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(A) Typical time course of change in pressure inside a mature ‘Sam’ sweet cherry fruit during a measurement of the whole fruit pressure () using the fruit pressure probe (FPP). A glass capillary is inserted into the fruit ≈5 mm below the epidermis at 0.5 min. On insertion, the pressure instantaneously increases. After a transient peak, the pressure equilibrates at ≈2.5 min. The equilibrium pressure as indicated by the arrow represents the . At ≈3.5 min, the capillary is withdrawn from the fruit and the pressure equilibrates again with the ambient pressure. (B) Change of of mature ‘Sam’ and ‘Samba’ sweet cherry as depth of insertion increases. The insertion depth represents the distance between the tip of the capillary of the FPP and the fruit surface. The regression equations were: (kilopascals) = 5.28 (± 1.88) + 0.73 (± 0.27) depth (millimeters), r² = 0.22, P = 0.01, n = 29 for ‘Sam’ and (kilopascals) = 6.73 (± 2.54) + 0.84 (± 0.35) depth (millimeters), r² = 0.19, P = 0.02, n = 27 for ‘Samba’. (C) Osmotic potential () and calculated water potential () for mature ‘Sam’ and ‘Samba’ at varying depth. Data for were redrawn from B. The was calculated as the sum of of expressed juice plus the for ‘Sam’ and ‘Samba’, respectively. Note that as a result of the very low , the symbols for and are superimposed in both cultivars.

Citation: Journal of the American Society for Horticultural Science 139, 1; 10.21273/JASHS.139.1.3

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Occasionally, the capillaries of the CPP or FPP became plugged or the seal between the capillary and cell wall (CPP) or between the capillary and fruit skin (FPP) became leaky. Both occurrences resulted in an obviously erroneous result. Plugging was easily recognized by a rapid pressure increase, whereas leakage was easily recognized by a continuing pressure decrease. Any such data were excluded from our analyses. After a pressure measurement, the CPP or FPP was withdrawn and this was always accompanied by a decrease in pressure to that of the ambient air recorded just before capillary insertion. The occasional absence of a pressure decrease on withdrawal was also indicative of plugging.

The and were quantified in sweet cherry and in selected drupe and berry crops using the CPP and FPP as described previously. The number of replications ranged from 12 to 46 successful insertions per species (mean 26 insertions) for determining by CPP and from six to 30 per species (mean 14 insertions) for determining by FPP.

The possibility of a radial gradient of within a sweet cherry was investigated in mature fruit of ‘Sam’ and ‘Samba’. The FPP was inserted at stepwise depth increments. The number of replications was 10. To relate to the and the water potential of the fruit ), a radial block of tissue (cross-section ≈2 × 2 mm, length ≈11 mm) extending from pit (endocarp) to skin (exocarp) was excised from the same batch of fruit. This block was divided transversely into five roughly equal parts. Each part was immediately crushed onto the sample holder of a water vapor pressure osmometer (VAPRO^® 5520; Wescor, Logan, UT) and the of its juice determined. In analogy to the cell’s water potential equation (Taiz and Zeiger, 1991), the was calculated from = + .

Quantifying by VPO.

The was also quantified using VPO (VAPRO^® 5520) and tissue discs excised from the sweet cherry mesocarp. Briefly, cylinders of mesocarp (diameter 8 mm) were excised from the cheeks of mature ‘Kordia’, ‘Sweetheart’, and ‘Staccato’ sweet cherries using a medical biopsy punch (BP-80F; Kai Industries, Gifu, Japan). Discs (≈2 mm thick) were cut from these cylinders with a razor blade. These were gently blotted using soft tissue paper (7216; Kimberly-Clark, Reigate, U.K.) and placed within 60 s of excision on the circular sample holder (9.5 mm diameter, 4.5 mm deep) of the osmometer. The water potential () was read 15 min after closure of the sample chamber when equilibrium reading had been reached. Next, the sample chamber was reopened, the cells destroyed by crushing with a glass rod, the chamber reclosed, and remeasured. The water potential reading thus obtained represented the after release of the component. Preliminary experiments established that there was no difference between a measured with (–3449 ± 118 kPa) and without cell walls (–3464 ± 123 kPa for juice only). Therefore, the results reported here are those determined for crushed discs including the cell walls.

Initial experiments were carried out to establish the time course of equilibration within the osmometer chamber of the water vapor pressure above the sample with the sample’s . The number of sample replications was five. Thereafter, and were quantified as described previously and was calculated for mature ‘Kordia’, ‘Sweetheart’, and ‘Staccato’ cherries using 10 fruit replications per cultivar.

Quantifying using the CP.

The CP as described by Bernstein and Lustig (1981) was used to quantify the of sweet cherry fruit. Briefly, a fruit was positioned between two glass plates in a universal material testing machine (Z 0.5; Zwick Roell, Ulm, Germany) equipped with a 500-N-force transducer (KAF-TC; Zwick Roell). The lower pressure plate was custom-made from translucent etched glass such that the aplanation area of the fruit could be photographed through it from below using a digital camera (Altra 20; Olympus Europa, Hamburg, Germany). To enhance contrast, the fruit surface was smeared with a thin coat of silicone grease. Unless specified otherwise, the cheek of the fruit opposite the suture faced the camera. Before initiation of the test, the compression plate was brought into contact with the fruit by applying a force of 0.05 N. The was determined by stepwise loading of the fruit with increasing force. After each force step, the position of the compression plates was held constant and a gradual decrease in force was observed as a result of gradual deformation (viscoelastic and/or plastic) of the fruit, which was followed over time until an asymptote was approached. Digital photographs were taken at selected time intervals and the aplanation areas between fruit and plate quantified by image analysis (Software CellˆP; Olympus Soft Imaging Solution, Münster, Germany). The (megapascals) was calculated as the slope of a linear regression line fitted through a plot of force, F (Newtons) vs. aplanation area A (square millimeters).

The time course of change in force, aplanation area, and was established in mature fruit of ‘Sam’. Individual fruit were loaded stepwise with 0, 1, 2, 4, 6, and 8 N and the changes in force vs. aplanation area at each force were followed over a 10-min period. The was calculated as described previously. The number of fruit replications was five.

The effect of orientation of the sweet cherry fruit during the test on was studied. Orientation was varied by placing mature ‘Sam’ fruit between the plates in the following orientations: suture up, suture down, or suture to the side (in all three orientations, the fruit’s pedicel/stylar-scar axis was horizontal). Compression tests were performed by stepwise increasing force to 0.5, 1, 2, 4, and 6 N. Aplanation area was recorded 5 min after applying each force when an approximate equilibrium had been reached. The number of fruit replications was seven.

Effects of water uptake or water loss on .

The effect of water uptake on was established in mature ‘Sweetheart’ sweet cherries using the FPP. Briefly, pedicels were cut flush with the receptacle and the fruit incubated in deionized water for up to 7.5 h. There was no silicone sealant applied to the pedicel fruit juncture. Water uptake was determined as the increment in fruit mass during incubation (fruit mass after, minus before) (Weichert et al., 2004). After water uptake, was quantified by FPP as described previously. The experiment was carried out on a total of 29 fruit.

The effect of water loss and of water uptake on was studied in mature ‘Regina’. The pedicels were cut flush with the receptacle. The cut surface of the pedicel and the remaining gap between the receptacle and fruit, subsequently referred to as the pedicel/fruit junction, was sealed with silicone sealant (3140 RTV coating; Dow Corning, Midland, MI) to exclude any water loss/gain around the pedicel/fruit junction (Beyer et al., 2002). After curing at ambient temperature for a minimum of 3 h, fruit were incubated above dry silica for 0, 24, 48, and 96 h. Water loss resulting from fruit transpiration was quantified gravimetrically. The was quantified on a subsample (n = 59) of fruit using the FPP. A second subsample of fruit (n = 55) was subjected to water-uptake experiments after the transpiration step. This fruit was incubated in deionized water. Water uptake was determined gravimetrically after immersion for 0, 0.75, and 1.5 h followed by careful blotting. Rates of uptake were calculated on an individual fruit basis from the slope of a regression line of a plot of cumulative fruit mass vs. time. Coefficients of determination averaged r² = 0.99.

Data analysis.

Data were subjected to analysis of variance (ANOVA). ANOVA, multiple comparisons of means, correlation, and regression analyses were carried out using Proc CORR, Proc GLM, and Proc REG (SAS Version 9.1.3; SAS Institute, Cary, NC). Data in tables and in Figures 2C, 3, and 4D are presented as means ± se. In all other figures, symbols represent data from individual fruit.

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Time course of equilibration of excised mesocarp discs (main graph) and of juice extracted from the discs (inset) in a water vapor pressure osmometer. At time zero, the sample chamber was closed and the change in apparent osmolality beginning at 80 s monitored over time. The mesocarp discs were excised and the juice extracted from ‘Kordia’ sweet cherry fruit. The arrows indicate the time required for equilibrating samples. At these times, the osmolality reading represented the water potential of the mesocarp disc () and the osmotic potential of the juice (inset; ).

Citation: Journal of the American Society for Horticultural Science 139, 1; 10.21273/JASHS.139.1.3

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Representative time courses of change in force (A) and aplanation area (B) when loading a mature ‘Sam’ sweet cherry fruit using a universal material testing machine. Immediately after reaching the preset force threshold of 1, 4, and 8 N, the position of the compression plates were held constant and the decrease in force and the increase in aplanation area monitored over time. (C) Relationship between force and aplanation area within 50 s of reaching the preset force level, between 100 and 200 s, and more than 500 s. The slope of the linear regression line fitted through these relationships equals the pressure inside an intact sweet cherry fruit (). (D) Time course of change in pressure inside the fruit during the compression plate test. The arrow indicates the pressure inside the fruit at equilibrium ().

Citation: Journal of the American Society for Horticultural Science 139, 1; 10.21273/JASHS.139.1.3

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Determining by CPP and VPO.

When the capillary of the CPP was inserted into the outer mesocarp of mature sweet cherry fruit, the pressure signal usually remained constant while some cell content was forced into the capillary by cell turgor (Fig. 1). Under the horizontal-axis microscope, a meniscus was visible between the red-colored cell contents and the silicone oil in the capillary. This meniscus is indicative of a successful insertion. Approximately 10 to 15 s after insertion, the meniscus was pushed back toward its initial position at the tip of the capillary by displacing a volume of silicone oil using the CPP’s micrometer screw. The push back was associated with an increase in the CPP system pressure and re-established the initial volume and pressure () of the punctured mesocarp cell (Fig. 1). Thereafter, the system pressure gradually decreased and approached an asymptote at a slightly lower level. A measurement of was judged successful if the following two conditions were met: 1) repressurizing the cell during the push back must increase the system pressure signal, which must equilibrate (asymptote) at an elevated level over a period of typically 1 to 2 min (Fig. 1); and 2) when the capillary was later withdrawn from the fruit, the CPP system pressure must drop to the same level as immediately before insertion. Occasional plugging of the capillary was detectable as a departure from this regular pattern. Such measurements were excluded from the analyses. Outer mesocarp values averaged 28.1 kPa in ‘Samba’ and 17.5 kPa in ‘Sam’ (Table 1). Meanwhile, of european plums, grapes, and tomatoes was somewhat higher (Table 1). In blueberries, nine of the 12 insertions yielded a value of 0 kPa. The remaining three measurements averaged 34.9 kPa, giving an overall average value of 8.7 kPa (Table 1).

Table 1.

Turgor of cells of the outer mesocarp and pressure inside a fruit () of selected drupe and berry fruit.^z

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When placing excised tissue blocks in the sample chamber of a VPO, the apparent osmolality increased with time, reaching an equilibrium ≈6 min after closure of the sample chamber (Fig. 3). From this time onward, the readout remained constant and the osmolality displayed corresponded reliably to the . Juice extracted from the tissue samples equilibrated within 80 s, yielding an estimate of the within the standard equilibration time of the instrument (Fig. 3, inset). Across the three sweet cherry cultivars, the indirect measurement, estimated by subtracting from , averaged 50 kPa (Table 2).

Table 2.

Water potential , osmotic potential (), and calculated cell turgor () of mesocarp discs excised from mature sweet cherry fruit.^z

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Determining by FPP and CP.

Inserting the capillary of the FPP in the mesocarp of sweet cherry and other drupe and berry fruit yielded changes of pressure with time, similar to those with the CPP (Fig. 2A). Usually an equilibrium value of was attained within 3 min. In fruit of nearly all species, an oil/water meniscus was not detectable. The exception was tomato, in which a meniscus was visible between the aqueous juice of the fruit and the silicone oil in the capillary. This meniscus was pushed back as described previously by turning the micrometer plunger. On withdrawal of the capillary from the fruit, the pressure declined to the level just before insertion indicating that there was no plugging of the capillary. Across cultivars and species, the values of as determined by FPP were of the same order of magnitude as those of by CPP or VPO but values were consistently lower (Table 1).

Increasing the depth from 1.1 to 9.9 mm within the ‘Sam’ and ‘Samba’ sweet cherry mesocarps to which the FPP was inserted slightly increased the measured value in both cultivars, but only by ≈6.5 and 7.4 kPa, respectively (Fig. 2B).

There was no detectable radial gradient in measured in the mesocarp of either ‘Sam’ or ‘Samba’ sweet cherry fruit and, hence, in the calculated values of . Because the averaged only 0.3% and 0.5% of the of the juice of ‘Sam’ and ‘Samba’ fruit, respectively, we can infer that the value of must have equaled (or been very close to) that of Ψ. Furthermore, when plotting , , and on the same y-scaling, the values of and were more or less superimposed for any cultivar or species and the gradient in was not discernable (Fig. 2C).

The compression plate test for determining yielded the relationships shown in Figure 4A–D. When applying a 1-, 4-, or 8-N force to the fruit and holding the position of the compression plates constant after peak force was reached, the force decreased exponentially with time (Fig. 4A), whereas the aplanation area of the fruit increased approaching an asymptote (Fig. 4B). The changes in force and aplanation area during the hold periods were larger at 8 N than at 4 or 1 N (Fig. 4A–B). Plotting pairs of values for force vs. aplanation area yielded essentially linear relationships (mean r² = 0.891, P = 0.001) in which the slopes depended on the elapsed time of the measurement after reaching peak force (Fig. 4B–C). The decrease in slope with increasing time resulted from viscoelastic and/or plastic deformation of the fruit as indexed by the decrease in force and the increase in aplanation area (Fig. 4A–C). Plotting this slope vs. the time for deformation revealed that within ≈3 min of the fruit being loaded, the slope of the force/area relationship had reached a steady state and the ratio of force/area equaled (Fig. 4D).

The determined by CP depended on how the fruit was aligned relative to the camera (and thus to the compressive force) (Table 3). The value was larger when the cheek opposite to the suture faced the camera and lowest when the suture faced the camera (Table 3). Orienting the fruit with the suture sideways (i.e., at 90° to the force axis) resulted in an intermediate .

Table 3.

Effect of orientation of sweet cherry on the pressure inside the fruit () as determined by the compression plate test.

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Effect of water uptake and loss on .

Incubating sweet cherry fruit in deionized water increased their mass at a rate of 26.0 (± 1.3) mg·h⁻¹ (r² = 0.94, P = 0.001) for up to 7 h (Fig. 5A). There was no significant relationship between the amount of water uptake and [ (kilopascals) = 10.5 (± 0.9) – 0.01 (± 0.01) × uptake (milligrams per fruit); r² = 0.04, P = 0.31 (Fig. 5B)].

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(A) Time course of water uptake of mature ‘Sweetheart’ sweet cherry. (B). Effect of water uptake on the pressure inside the fruit () as determined by the fruit pressure probe (FPP). Fruit were allowed to take up water for up to 7 h, the amount of water taken up quantified gravimetrically, and the determined by FPP.

Citation: Journal of the American Society for Horticultural Science 139, 1; 10.21273/JASHS.139.1.3

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Similarly, the amount of water transpired increased linearly with time at an average rate of 15.2 (± 0.5) mg·h⁻¹ (r² = 0.94, P = 0.001; Fig. 6A) but, again, there was no significant relationship between the amount of water lost and [ (kilopascals) = 13.9 (± 1.3) – 1.15 (± 1.69) × transpiration (grams per fruit); r² = 0.01, P = 0.50 (Fig. 6B)]. Fruit that had transpired more water had lower rates of water uptake than fruit that had transpired less indicating that the rate of water uptake and the amount of water transpired were weakly and negatively correlated [r = –0.43, P = 0.0012 (Fig. 6C)].

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(A) Time course of transpiration of mature ‘Regina’ sweet cherry. (B). Whole fruit pressure () as affected by transpiration. The was quantified using the fruit pressure probe (FPP). (C) Relationship between the amount of water lost during transpiration and subsequent water uptake. Here, fruit were allowed to transpire and the amount of transpiration quantified gravimetrically. Subsequently, the same fruit were incubated in water and the amount of water taken up quantified gravimetrically in a water uptake assay.

Citation: Journal of the American Society for Horticultural Science 139, 1; 10.21273/JASHS.139.1.3

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Comparing and with published data.

Published data for range from 1.5 to 2.1 MPa in sweet cherry (Andersen and Richardson, 1982). Measham et al. (2009) determined critical turgor pressures at maturity for nine sweet cherry cultivars; these averaged 2.8 MPa (range 2.0 to 3.8 MPa). Knoche et al. (2004) calculated turgor pressures ranging from 0.55 to 0.77 MPa in mature sweet cherry. Somewhat lower has been reported for other cracking-susceptible fruit crops such as grapes [0.3 MPa (Matthews et al., 1987)], gooseberries (0.7 MPa), black currants (0.5 MPa), and gooseberry × black currant hybrids [Ribes nidigrolaria B. (0.6 MPa)] (Khanal et al., 2011). All these estimates share two commonalities. First, all the values reported are strictly apparent values of because they were not measured directly but were calculated by a difference from measurements of whole-fruit water potential and of the of the fruit’s expressed juice. Second, these values are surprisingly high. In fact, most are almost 10 times higher than the pressure in a car tire (car tires are usually inflated to ≈0.2 MPa). Given this benchmark, the fruit pressure they suggest (≈2 MPa) should be seen as counterintuitive bearing in mind the soft “feel” of a sweet cherry compared with the hard feel of a car tire. If the car tire were to be overinflated to 10 times the recommended pressure (from 0.2 to 2 MPa), it would “feel” like steel! To our knowledge, there have been no direct measurements of or of in sweet cherry fruit. In grapes, which have a similar architecture, the and values have been quantified using pressure probes (Lang and Düring, 1990; Thomas et al., 2006, 2008) and using compression plates (Bernstein and Lustig, 1981). These techniques measure and/or directly. The post-veraison and values reported by these authors were always lower than 0.1 MPa at maturity (Bernstein and Lustig, 1981, 1985; Lang and Düring, 1990; Thomas et al., 2006, 2008). These values are very much lower than the indirect values referred to previously and are moreover consistent not only with the values we obtained here but also with the soft feel of the fruit. One possible reason why some indirect estimates of fruit pressure are so high could stem from confusion over the property measured by a pressure bomb (Scholander et al., 1965). A pressure bomb measures not apoplast water potential, but apoplast pressure. These two are numerically the same only when apoplast Ψ_Π is close to zero. The pressure bomb is most commonly used to measure apoplast pressure in transpiring leaves. Here, this precondition is usually met and thus the common (but slightly careless) terminology has arisen where we talk of measuring leaf water potential with a pressure bomb. This being the case, some of the indirect fruit pressure estimates are probably in error.

Possible reasons for low and .

Because the cuticle is an effective barrier to either liquid- (ingress) or vapor-phase (egress) water transport through the fruit surface, we expect the tissues inside the fruit and hence the fruit’s apo- and symplasts to be in reasonably close water potential equilibrium, = . Under these conditions, the sums of the osmotic and pressure components of the two compartments can be equated as + = + . For the large, thin-walled cells of the mesocarp, the volume fraction of the symplast very greatly exceeds that of the apoplast so the measured of the expressed juice will likely be equivalent to . Because the (CPP, VPO) and the (FPP, CP) are very close to atmospheric pressure, we can infer that the is numerically close to the and that is similar to . This being the case, it seems likely that osmotically active substances have accumulated in the apoplast. To our knowledge, there is no direct evidence for an accumulation of significant in sweet cherries. However, in post-veraison grapes, solutes were shown to accumulate in the apoplast and essentially eliminate the (Matthews and Shackel, 2005; Thomas et al., 2009; Wada et al., 2008, 2009). Theoretically, such solutes may result from leakage of the symplast caused by a loss of compartmentation or decreased reflection coefficients of plasma membranes (Lang and Düring, 1991) or from rupture of plasma membranes resulting from the bursting of individual cells (Tilbrook and Tyerman, 2008). Alternatively, apoplastic solutes may result from a lack of loading of solutes that had been unloaded from the phloem into the sweet cherry apoplast as was proposed for stimulating phloem transport in the turgor-regulated phloem translocation hypothesis (Lang, 1983) or as was demonstrated for water-cored apple (Malus ×domestica Borkh.) tissue (Gao et al., 2005).

Lack of response of to water uptake and transpiration.

The absence of a significant effect of water uptake on is surprising, particularly because 1) water uptakes of up to 0.2 mL result in significant cracking in this (M. Knoche, unpublished data) and earlier studies (Christensen, 1996; Weichert et al., 2004); and 2) based on current theories, cracking is thought to be related to increased (Measham et al., 2009; Sekse, 1995; Sekse et al., 2005).

A reasonable explanation accounting for water uptake without an associated increase in is that the skin suffers an increase in strain without an associated increase in stress. Recent studies by Grimm et al. (2012, 2013) demonstrate that the strain in the exocarp of mature sweet cherry fruit is largely reversible and is released in a time-dependent manner after excision. This behavior is referred to as viscoelasticity (Niklas, 1992; Vincent, 1990) and is a common characteristic of cell-wall composites. Chanliaud et al. (2002) presented evidence that the hemicellulose fraction of the cell-wall polymers is likely responsible for viscoelasticity. In any system, it is inevitable that under conditions of increasing strain, there will be a tendency for a build up of stress. Because there was no detectable increase in with water uptake, we must infer that stress release occurs almost instantaneously, i.e., that the stress-release processes are rapid relative to the rates of strain associated with water uptake.

The absence of a significant would also account for the lack of a significant relationship between the rate of transpiration and that of water uptake. This inference is based on the following argument. The maximum amount of water transpired over 94 h averaged ≈1.4 g per fruit. This loss will have caused the osmotic concentration of the juice and, hence, the driving force for water uptake to increase by ≈15% (osmolality will have increased by 178 mmol·kg⁻¹ or 0.44 MPa). This driving force increase will have been offset by a fruit surface area decrease of ≈–8.9% (–2.1 cm²). We can reasonably expect that the net result of the transpirational water loss would be an increase in water uptake rate of only 4.8%. With fruit:fruit variability, this change would not have been detectable in our gravimetric water transport experiments.

Comparing pressure probe, compression plate, and water vapor osmometry for quantifying and .

Despite of their quite different measurement principles, the pressure values obtained using these three techniques were remarkably similar. Nevertheless, some small but consistent differences were observed that deserve comment.

The CPP and the FPP are the most widely used direct measurement techniques. Although their principles of measurement are identical, some differences are worth noting. The fine capillary tip (30 to 60 μm diameter) of the CPP allows individual cells to be punctured. Thus, the CPP quantifies the pressure (relative to atmospheric pressure) of the symplast of a single cell usually defined as cell turgor (here, ). Because the epidermal and hypodermal cells of mature sweet cherries are too small (less than 50 μm diameter) to be successfully punctured, measurements were restricted to the much larger cells (100 to 200 μm diameter) of the outer parenchyma at depths of between 200 and 400 μm below the fruit surface. In contrast, the capillary of the FPP is significantly wider at the tip (150 to 250 μm diameter) and hence many of the large (100 to 200 μm diameter), thin-walled cells of the outer parenchyma are ruptured. This results in a general mixing of the symplast (relatively large) and apoplast (relatively small) volumes in the vicinity of the probe aperture. The FPP measures the resultant pressure of this mixture of ruptured cells: protoplasts, cell walls, intercellular fluids. We refer to the pressure at this site as the . Based on the theory of shells (Considine and Brown, 1981), this (megapascals) resulted from the stress (σ in Newtons per square millimeter) caused by the elastic strain of the fruit skin according to = where t (millimeters) represented the thickness of the structural shell and r (millimeters) the radius of the sphere.

Compared with the FPP, the CP uses quite different physical principles to measure . Increasing, bilateral compressive forces are applied stepwise to the fruit and the aplanation area for each force step is quantified. In contrast to CPP and FPP, the method is much easier to operate. There are no potential artifacts such as plugging of a probe tip or leakage around the shoulder of the probe like with CPP or FPP. The values recorded by CP were of the same order of magnitude as the FPP ones but were nevertheless slightly higher. The reasons for this difference are unknown. Several factors may be involved. First, CP tests are based on the assumption that the fruit resembles a liquid-filled vessel where the incompressible liquid inside is held under pressure by a stressed skin. Although this model may be applicable to sweet cherry fruit over long time scales (greater than 1 d), it is unlikely to hold for the shorter time scales (less than 1 h) in our CP experiments. Over these shorter time scales, the tissue offers some resistance to deformation thereby absorbing some of the force applied. This would result in a slight overestimation of by the CP compared with one determined by the FPP. Second, mature sweet cherry fruit are not perfect spheres and this may be why measurements of by CP depend to some extent on the fruit’s shape and its orientation on the plate. Third, the analysis of the CP data assumes structural homogeneity of the skin, but skin extensibility and, possibly, flesh deformability of cherry fruit could well vary between regions of the fruit. For example, heterogeneity may arise from the presence of pedicel, stylar scar, suture, or from vascular bundles within the mesocarp. These factors may have contributed to the small deviations between the data by CP and by FPP. However, it is important to notice that although these deviations are significant, they are also very small. So it is fair to conclude that the CP represents an accurate and also a relatively robust and straightforward method for determining .

The VPO method differs from all the others in that it is indirect. It yields a calculated estimate for that is obtained by difference from an evaluation of and of . On excision, sweet cherry tissue relaxes in a time-dependent manner. Grimm et al. (2012) reported a half time of relaxation for skin segments of ≈2.7 min. In our experiment, the excised tissue disc was introduced within 1 min into the sampling chamber and equilibrated for 15 min before the reading was taken. During this time, the relaxation of the tissue would be essentially complete. Also, we would expect relaxation of the thin-walled parenchyma disc to be more rapid than that of the collenchymatous fruit skin segments used by Grimm et al. (2012). Consequently, any error resulting from tissue tension would have been small.