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Grape berries can lose weight and shrivel at advancedstages of maturity. Berries of Vitis vinifera L. cv. Shiraz areunusual in that the weight loss can occur well before theberries are suitable for harvesting, even under adequate ir-rigation (McCarthy 1999). This weight loss can begin 80 to115 days after flowering, depending on season, and canreach up to 30% of maximum weight prior to harvesting.Nearly all of the weight loss (90%) is in the form of watervapor, while loss in dry weight could be accounted for byberry respiration (Rogiers et al. 2000). Impeded vascularflow from the vine into the berry during the later growthstage has been hypothesized to play a role in shriveling atsuboptimal Brix (McCarthy and Coombe 1999). Unusuallyhigh berry transpiration rates may be another physiologicalcause for this condition, leading to excessive water loss(Düring et al. 1987, Lang and Thorpe 1989). If influx of wa-ter to the berry through the pedicel cannot compensate forwater loss through the cuticle, then the shriveling symp-toms often observed in Shiraz may result.

Grape Berry cv. Shiraz Epicuticular Wax and Transpirationduring Ripening and Preharvest Weight Loss

Suzy Y. Rogiers,1,2* Jo M. Hatfield,1,2 V. Gunta Jaudzems,3

Rosemary G. White,4 and Markus Keller2,5

1Cooperative Research Centre for Viticulture, PO Box 154, Glen Osmond, SA 5064, Australia; 2NationalWine and Grape Industry Centre, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678,Australia; 3Monash Micro Imaging, School of Biological Sciences, Monash University, PO Box 18,Clayton, Victoria 3800, Australia; 4CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia;and 5Washington State University, Irrigated Agriculture Research and Extension Center, Prosser, WA99350 USA.

*Corresponding author [Fax: +61 2 6933 2107, email suzy.rogiers@agric.nsw.gov.au]

Acknowledgments: This research was supported by the Commonwealth Cooperative Research CentreProgram and conducted through the CRC for Viticulture with support from Australia’s grapegrowers andwinemakers through their investment body the Grape and Wine Research and Development Corporation,with matching funds from the federal government. We thank Paul Kriedemann for helpful discussionsduring the course of this study. Technical assistance by Robert Lamont is also gratefully acknowledged.Thanks to the Charles Sturt Winery for permitting fruit sampling and to the vineyard staff for vinemaintenance.

Manuscript submitted October 2003; revised December 2003, February 2004

Copyright © 2004 by the American Society for Enology and Viticulture. All rights reserved.

Abstract: Preharvest weight loss of Vitis vinifera L. cv. Shiraz berries may be the result of cuticle disruption leadingto high transpiration rates relative to earlier stages of ripening. Scanning electron microscopy showed very fewbut functional stomata on young berries and wax-filled stomata on older berries and, aside from slight cracks alongthe stomatal protuberance, did not reveal any fissures in the surface of berries that may lead to increased transpirationrates. Preveraison berry epicuticular wax platelets were defined and intricate, while postveraison and shriveled berrysurfaces had large areas of amorphous waxes. Postveraison, the area of amorphous wax relative to intricate waxwas not correlated with berry age or degree of shrivel. Extraction of surface waxes revealed that total wax on asurface-area basis decreased during veraison then remained stable as the berries ripened and entered the weight-loss phase. Berry transpiration, estimated from loss of fresh weight of detached berries over time, during this finalshriveled phase was 16% of the preveraison rate on a per berry basis. Nevertheless, berry transpiration could accountfor an average 15 mg loss in fresh weight per berry per day. We conclude that weight loss during late ripening ofShiraz berries was not the result of cuticle disruption or high transpiration rates alone. It is hypothesized thatdecreased vascular flow of water into the berry combined with continued transpiration leads to the weight loss.

Key words: Vitis vinifera, berry, shrivel, transpiration

The cuticular membrane covers all aboveground parts ofterrestrial plants, including their fruits. This membrane con-sists of a polymer matrix (cutin), polysaccharides, and sol-vent-soluble lipids (cuticular waxes) (Holloway 1982). Thecuticle controls water movement between the epidermalcells and the ambient atmosphere (Riederer and Schreiber2001). When present, stomata are the preferred sites of wa-ter loss. In grape berries, however, their frequency is evenlower than that of leaves of crassulacean acid metabolismplants (Blanke and Leyhe 1987), and it is likely that the cu-ticle is important in controlling water loss. The structuralarrangement of the wax, together with its chemistry, con-trols the water movement from berries (Chambers andPossingham 1963, Possingham et al. 1967). The structure ofepicuticular waxes changes with fruit age. Although nor-mally crystalline, the wax material is rather soft and can bealtered or removed by the impact of rain, abrasion fromwind-blown particles, or contact with other berries, leaves,or other adjacent objects. On some leaf surfaces investi-gated, the crystals have appeared to degrade slowly overtime, fusing into amorphous masses and eventually into acontinuous layer on top of the normal, uninterrupted waxlayer (Reicosky and Hanover 1976, Grill et al. 1987).

Reynhardt and Riederer (1991) proposed a model of thephysical structure of the epicuticular wax layer, describingit as a matrix composed of highly ordered crystalline anddisordered amorphous regions. Reduced diffusional dynam-ics in the crystalline regions of the wax barrier make thempractically impermeable. Thus, transport across the wax bar-rier may be expected to occur only through the amorphous re-gions. The physical state of the wax layer, more than its chemi-cal composition, may determine the transport properties of the

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cuticle. It is therefore important to investigate the relativedistribution of crystalline and amorphous regions on theShiraz berry and to determine if a potential transition fromcrystalline to amorphous wax is related to the shrivel phe-nomenon. Moreover, cuticle disruption in fruits can be dueto microbial pathogens or sun exposure and extreme tem-peratures. Cracks in the cuticle of expanding berries havealso been reported for some varieties (Percival et al. 1993,Comménil et al. 1997). The presence of these cracks wouldprovide open wounds, and once damaged, the berry may bemore susceptible to high rates of water loss.

The objectives of the present work were to examine thesurface of the Shiraz berry during late ripening and shrivel-ing for evidence of cuticular disruption and to correlateproperties of the waxy cuticle with transpiration of indi-vidual berries during development.

Materials and Methods

Berry weight. Five bunches of berries were harvested atrandom from field-grown Shiraz vines twice weekly fromflowering to harvest over the 1999 to 2000 and the 2001 to2002 seasons. These 11-year-old, own-rooted vines werefrom clone PT23/N/Griffith and were located at Charles SturtUniversity, Wagga Wagga, NSW, Australia. Fifty-berry sam-ples were taken at random from each bunch and weighed.

Epicuticular wax extraction. Fifty berries from threebunches of each sampling date of the 1999 to 2000 seasonwere weighed and briefly rinsed in water to remove dirt anddust. They were dried on filter paper and then dipped inchloroform three times in separate vials (30 sec, 10 sec, 1sec). Chloroform extracts were combined in tarred vials andallowed to evaporate at 22°C under nitrogen flux overnightand then dried at 40°C for 5 to 7 days until constant weight.Water-soluble exudates were partitioned from the waxes bydissolving in water (5 mL), then chloroform (5 mL). The wa-ter layer was removed with a pipette and the chloroformlayer was dried as described above.

Scanning electron microscopy. Shiraz berries weresampled during the 1999 to 2000 season at random from thefield vines described above, taking care not to make contactwith the surface of the berry. Vitis vinifera L. cvs. CabernetSauvignon and Chardonnay berries were also sampled fromthe same vineyard. Some berries were plunged into liquidnitrogen, and 1-cm2 skin sections were freeze-dried, at-tached to aluminum stubs, and sputter-coated with gold at0.05 mbar and 25 mA for 3 min prior to scanning electronmicroscopy (SEM). In a second method, a 1-cm2 slice wascut from each berry, attached to a flat brass stub, quick-fro-zen at approximately -170°C on the cold stage of a JEOL6400 SEM (JEOL Australasia Pty Ltd, Sydney, Australia),gold-coated, and then observed.

Transpiration. Berries of five developmental stages (sixreplicates) from preveraison to shriveled were harvested atrandom from the field vines described above during the2001 to 2002 season. Height and width of each berry were

measured with callipers, and surface area was estimatedaccording to the modified equation for a sphere: surfacearea = π(width)(height). Pedicels were dipped in paraffinwax to prevent water loss from this exposed surface. Berrieswere suspended in an incubator at 25°C with silica gel tomaintain a constant 23% relative humidity without air turbu-lence. Berries were weighed twice daily over three days, andtranspiration rates were calculated from the slope of the lin-ear regression of weight loss over time (the r2 of each slopewas ≥0.994 ), taking into account an average daily respira-tion rate of 1.9 mg CO2 per berry (Niimi and Torikata 1979).

Results

Berry weight. In the 1999 to 2000 season, gain in berryfresh weight was biphasal with a central transition point(Staudt et al. 1986, Blanke 1992) one week before veraison(67 days after flowering, DAF) (Figure 1A). Mean berryfresh weight reached a maximum at 95 DAF (1.5 g), and thendeclined by 20% to 1.2 g at 115 DAF, giving an averageweight loss of 15 mg per berry per day. In the 2001 to 2002

A

B

Figure 1 (A) Berry fresh weight (n = 5, 50 berries per sample) and (B)berry epicuticular wax amounts (n = 3, 50 berries per sample) duringdevelopment and ripening. Inset refers to mass of chloroform extractprior to partitioning with water to remove berry exudates (n = 3, 50berries per sample). Bars indicate standard error of the mean.

A

B

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season, veraison again occurred at 67 DAF and the berryweight maximum occurred at 106 DAF (1.6 g). Berrieswere harvested at 133 DAF when they weighed 1.2 g.Weight loss was 25%, with an average weight loss of 15mg per berry per day as in the 1999 to 2000 season.

Epicuticular wax. Mass of the primary chloroform ex-tract per unit berry surface area prior to water extractionwas 1.4 ± 0.1 µg mm-2 and did not change through devel-opment (Figure 1B). After removal of water-soluble berry-surface exudates from the chloroform extract, mass on asurface-area basis ranged between 1.1 and 1.2 µg mm-2

from 35 to 50 DAF, then declined by 90 DAF (berryweight maximum) to 17% of peak values (0.2 to 0.4 µg waxmm-2) (Figure 1B) and remained low through the weight-loss phase until berries were harvested at 110 DAF. Incontrast, the water-soluble exudates increased from ap-proximately 18% of the total surface exudates at 35 to 50DAF to 83% of exudates by 90 DAF.

Morphology of the grape berry. The morphology ofthe grape berry changed during fruit ontogeny and rip-ening. At anthesis the ovary was covered in cuticularridges (Figure 2A), which had spread out by 20 DAF(Figure 2B) and wax was present as grains (Figure 2C).Occasional protuberant stomates (on average less thanfive per berry) were also visible (Figure 2D). Stomateswere visible on the flower cap prior to bloom, on theovary, and on the preveraison berry. These stomateswere hidden once the epicuticular waxes covered theberry. One or two stomates could be seen on post-veraison berries, but they were mostly filled by the wax(data not shown). At 40 DAF the waxes formed well-de-fined plates with fringed edges and were so dense thatthe cuticle membrane underneath was no longer visible(Figure 2E, F, G). In the postveraison berry, the cuticleand epicuticular wax was approximately 2-µm thick (Fig-ure 2H). The defined vertical platelets also featured inthe postveraison and post-weight maximum berries (Fig-ure 3A, B, C) and were similar to those seen on CabernetSauvignon (Figure 3D) and Chardonnay (data notshown). From veraison onward, large areas of amorphouswax were also observed in all three varieties (Figure 3E).The top ends of the vertical platelets appeared to spreadinto umbrella-like horizontal plates that condensed toform a smooth crust (Figure 3F, G). These smooth areascovered from 10 to 80% of the berry and the area wasnot dependent on the age of the postveraison berry orthe degree of shrivel. However, sun-exposed berries hadlarger areas of the amorphous wax than shaded berries.The wax was also amorphous in those areas where ber-ries were in contact with other berries.

Cavities or fissures were not visible in plump, slightlyshriveled, or moderately shriveled Shiraz berries. How-ever, there were tiny cracks along some of the stomatalprotuberances in several of the postveraison berries.Moreover, very tightly shriveled berries also had cavi-ties in the surface (Figure 3H).

Figure 2 Scanning electron micrographs of grape berry surfaces at variousdevelopmental stages. (A) Cuticular ridges on the surface of Shiraz ovaryat anthesis. (B) Surface of Shiraz preveraison berry at 20 DAF. (C) Close-up of (B) showing cuticular ridges and rudimentary wax granules. (D) Stomateon berry surface at 20 DAF. Note the lack of wax granules near the opening.(E–H) Typical berry surface at 40 to 120 DAF featuring upright, intricatescalelike wax platelets. (H) Transverse section of the dermal and cuticlelayers of a postveraison berry; cuticle, including epicuticular wax layer, ~2µm thick. Bar in A, F, H = 10 µm; bar in B, D = 20 µm.; bar in C, G = 5 µm; barin E = 100 µm.

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Transpiration. From the preveraison to the post-veraison phases of berry development, transpiration ratesper unit surface area decreased to 19% of original rates(Table 1), which then decreased by another 3% duringthe shriveled phase. On a per berry basis, transpirationof shriveled berries was 16% of preveraison rates andwas equivalent to 15.2 mg berry-1 day-1.

Discussion

The epicuticular wax development on Shiraz berries inthis study was similar to that described earlier for othervarieties, for example, cvs. Thompson Seedless (Rosen-quist and Morrison 1988) and Palomino fino (Casado andHeredia 2001). Cuticular ridges were present at flowering,and these spread out and fused as the berry expanded.Rosenquist and Morrison (1988) suggested that the cu-ticular ridges function as a storage form of cuticular ma-terial, which spreads as the berry expands. The first epi-cuticular wax appeared as small grains and then formedinto intricate crystalline platelets 20 to 40 days after flow-ering. This was followed by the development of an amor-phous wax layer on top of the existing platelets post-veraison. The proportion of the berry surface that wascovered with amorphous wax was larger on sun-exposedberries than on shaded berries, perhaps related to thesubstantially higher temperature experienced by the ex-posed berries. Such berries often reach 15°C above ambi-ent temperature, whereas shaded berries are generallyclose to ambient temperature (Smart and Sinclair 1976,Spayd et al. 2002). The surface wax of Valencia orangefruit changed from upright plates to amorphous duringdevelopment, a change that occurred earlier for exposedfruit (El Otmani et al. 1989). There were extensive areas ofamorphous wax on postveraison berries in our study.However, shriveled berries did not have more extensiveamorphous wax than plump postveraison berries, sug-gesting that shriveling in Shiraz berries is not the resultof amorphous cuticular wax development.

Fissures on skins of berries may lead to increased tran-spiration rates. Fissures were observed in cvs. Optima(Percival et al. 1993) and Pinot noir (Comménil et al. 1997)skins at maturity. Our investigation revealed no fissuresalong the surfaces of postveraison, mildly shriveled, ormoderately shriveled Shiraz berries. Cabernet franc ber-ries are relatively small, and like Shiraz berries in thisstudy, these did not show any fissures traversing the cu-ticle (Percival et al. 1993). Cuticles of varieties with largerberries may be more susceptible to fissures because thereis lack of adequate mechanical support (Percival et al.1993). In some varieties small cracks can develop at thebase of stomatal protuberances during veraison (Blankeet al. 1999). We also observed these cracks on some post-veraison berry stomata, but, as this phenomenon occursin many varieties, it is unlikely that it would lead to theshriveling that is unique to Shiraz. The cavities in the se-

Figure 3 Scanning electron micrographs of Shiraz grape berry surfaces.(A) Moderately shriveled berry at 100 DAF. (B) Close-up of (A) showingintricate wax platelets. (C) Intricate wax platelets of severely shriveledberries at 120 DAF; note fungal hyphae. (D) Wax platelets of CabernetSauvignon postveraison berry, similar to that of Shiraz. (E) Amorphouswax formation seen on portions of Shiraz, Chardonnay, and CabernetSauvignon berries 60 to 120 DAF. These waxes can cover up to 80% ofthe berry. (F, G) Close-up of (E) showing formation of amorphous waxeson top of platelets. (H) Cavities in the surface of tightly shriveled anddecaying berries (not present in mildly or moderately shriveled berries).Bar in A, E, H = 100 µm; bar in B, C, D = 10 µm; bar in F, G = 5 µm.

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verely shriveled berries were likely a by-product of shrivelas opposed to a cause of shrivel, as they were not apparentin earlier stages of shriveling.

In order to determine whether decreasing wax quantitiescontributed to Shiraz shrivel, chloroform was used to re-move the surface wax from the grape berries. The main com-ponent of berry wax is a hard wax, oleanic acid, whereassoft wax is a mixture of long-chain fatty acids, alcohols, al-dehydes, esters, and hydrocarbons (Radler and Horn 1965,Yamamura and Naito 1983, Comménil et al. 1997). For ex-ample, Yamamura and Naito (1983) found that Delawareberry surface wax was predominantly hard wax during allgrowth stages (87% at maturity). Since chloroform solubi-lized not only the waxes but also the other exudates on theberry surface, these exudates were removed from the chlo-roform extract by washing in water. The remaining chloro-form layer was then dried down to constant weight. Berryexudates consist of phenolic compounds and malic acid inimmature fruit, and as the fruits mature they contain sugars,malic acid, potassium, and sodium (Padgett and Morrison1990).

The total mass of primary chloroform extract remainedunchanged, on a surface area basis, throughout develop-ment. This result contrasts with the finding of Palliotti andCartechini (2001), who reported a substantial increase inchloroform-extracted wax (from 1 up to 5 µg mm-2) frompostflowering to postveraison Cabernet Sauvignon berries.This contrast is starkly enhanced by removal of the watersoluble extracts, which revealed a substantial decrease inwax, on a surface-area basis, from veraison until the berryweight maximum (1.2 to 0.3 µg wax mm-2). This may seemsurprising considering that the scanning electron micros-copy images indicated the postveraison development of anamorphous layer on top of the existing wax platelets. How-ever, it was observed in Pinot noir grapes that, despite theformation of this layer, cuticle thickness decreased as theberry expanded (Comménil et al. 1997). The density of thewax platelets may also decrease as the berry expands,which may result in an overall decrease in wax quantitiesper surface area.

As indicated above, in other varietiesthere appears to be no consistent corre-lation between berry development andwax thickness or quantity per surfacearea. For example, the wax of Delawaregrapes increased during the early pre-veraison stage (until 30 DAF) and thenremained constant thereafter (Yamamuraand Naito 1983). In Thompson Seedlessgrapes, however, no change in the totalwax (1 µg mm-2) during berry growth wasreported (Radler 1965), and an increasein the weight of wax per unit surface areaoccurred during development of PinotNoir grapes (Comménil et al. 1997). Ourresults for Shiraz suggest that the forma-

tion and deposition of epicuticular wax occurs early duringberry development and ceases before the central transitionpoint of berry growth. These conflicting reports on amountof berry surface wax may be due to differences in methodsof wax extraction. Furthermore, the length of the develop-mental period investigated and environmental factors suchas temperature, light exposure, relative humidity, and irriga-tion (Rosenquist and Morrison 1989) will have an effect ontrends.

One might intuitively assume that the amount of epicu-ticular wax will affect cuticular transpiration. In this study,however, there was no negative correlation between amountof wax and berry transpiration. Berry transpiration per sur-face area decreased from preveraison to postveraison andthen decreased even further during the shriveling phase ofripening. Similarly, transpiration of postveraison Müller-Thurgau (Leyhe and Blanke, 1989) and Cabernet Sauvignon(Greenspan et al. 1994, 1996) berries was less than that ofpreveraison berries. The composition of the cuticle and theepicuticular wax may be more important than its thicknessor density for determining transpiration rates. The physicalstructure of the wax, such as crystallinity, may also affecttranspiration rates (Reynhardt and Riederer 1991), but asthere was no correlation between area of amorphous waxand incidence of shrivel, other factors may be involved.

Loss of stomatal functioning may also contribute to thedecreased transpiration during ripening of grape berries(Palliotti and Cartechini 2001). Shortly after fruit set, the sto-mata were raised on a conical protuberance and appearedto be functional. An “aureole peristomatique” was first de-scribed for the grape berry by Bessis (1972), and its protu-berance was discovered and labeled “stomatal protuber-ance” by Blanke and Leyhe (1988). The very few stomata weobserved in postveraison berries in this study had beentransformed into nonfunctional lenticels and were primarilyfilled with wax. The continued decrease in berry transpira-tion after veraison is further evidence that shrivel is notcaused by fissures or cavities in the skin. Such physicaldamage would be expected to increase transpirational waterloss rather than to decrease it.

Table 1 Transpiration rates of five developmental stages of Shiraz berries sampled onthe same day. Loss in fresh weight of detached berries monitored over three days at25°C and 23% relative humidity (VPD = 2.45 x 10-3 MPa). Transpiration rate calculatedby subtracting a daily respiratory loss of 1.9 mg CO2/berry (Niimi and Torikata 1979)

as carbon from fresh weight loss. Values are means ± standard errors.

Berry maturity Soluble solidsa Transpiration rate Transpiration rate(Brix) (µmol m-2 s-1) (µmol berry-1 s-1)

Green (preveraison) —b 129 ± 3 0.062 ± 0.002Pink (50%) 22.6 ± 0.7 62 ± 2 0.030 ± 0.001Pink (100%) 22.6 ± 0.7 47 ± 2 0.023 ± 0.001Blue (postveraison) 31.2 ± 0.9 24 ± 1 0.0117 ± 0.0006Shriveled 44.0 ± 0.8 21 ± 1 0.0098 ± 0.0006

aSoluble solids readings taken after three days of transpiration measurements.bPreveraison berries did not contain adequate amounts of juice for a soluble solids assessment.

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Transpiration rates of shriveled berries were equal to therates of weight loss observed in field berries (on average15 mg water vapor berry-1 day-1). However, since transpira-tion rate decreased on both a surface-area and whole-berrybasis during ripening, it is unlikely that this alone would bethe cause for shrivel. In a growing postveraison berry(prior to the weight maximum), net water flow into the berrymust exceed net water loss. In a shriveling berry, the rate ofwater loss through transpiration was slightly less than thatof the growing postveraison berry, indicating that net waterflow into this shriveling berry must have been less thanthat of the growing postveraison berry. If transpirationrates had suddenly increased during shriveling, then tran-spiration alone could have accounted for the transforma-tion of a plump berry into a shriveled one. While Coombeand McCarthy (2000) have hypothesized that xylem flowinto the berry decreases at veraison and that phloem flow isdiscontinued at the weight maximum, earlier studies usingelement accumulation patterns indicated that xylem flowinto the berry is not necessarily completely impeded afterveraison (Ollat and Gaudillère 1996, Rogiers et al. 2000).Tracer dye studies pointed to a xylem disruption betweenthe brush region of the berry and the rest of the berry(Rogiers et al. 2001). However, this disruption does notnecessarily indicate a complete cessation of xylem ex-change between the vine and the berry since water couldpotentially flow into the berry through the xylem up to thebrush and then continue along symplastic routes (be-tween adjacent cells through plasmodesmata) from thebrush into the pulp and skin regions. Although not thoughtto be important after veraison (Greenspan et al. 1994),backflow has been reported in grapes (Lang and Thorpe1989) and may play a role in shrivel. Further elucidation ofthis phenomenon, together with data on rates of phloemflow into the berry, is required to better understand thephysiological cause of shriveling in Shiraz berries.

ConclusionThe objective of this study was to examine the role of

berry transpiration in Shiraz berry shrivel. Epicuticular waxamounts on a surface area basis declined after veraison,but did not decline further after the berry weight maximum.The epicuticular wax platelets were intricate or amorphous,with no correlation between the area of amorphous waxand incidence of shrivel. There were no fissures in thesurface of the cuticle that might lead to increased transpi-ration rates. Small cracks were visible along the stomatalprotuberance, but that was not likely associated withshrivel as the cracks were present in preweight maximumberries and are fairly common among other grape varieties.As fruits ripened and subsequently shriveled, there was adecline in transpiration on both a whole-berry and surface-area basis. Shriveling in Shiraz was not caused by a suddenspike in transpiration rates. Rather, we hypothesise that adecrease in net vascular flow into the berry together withcontinued transpiration leads to the weight loss.

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