Grape Berry Cv. Shiraz Epicuticular Wax and Transpiration During Ripening and Preharvest Weight Loss

Epicuticular Wax and Transpiration – 121

Grape Berry cv. Shiraz Epicuticular Wax and Transpiration during 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

Abstract: Preharvest weight loss of Vitis vinifera L. cv. Shiraz berries may be the result of cuticle disruption leading to high transpiration rates relative to earlier stages of ripening. Scanning electron microscopy showed very few but functional stomata on young berries and wax-filled stomata on older berries and, aside from slight cracks along the stomatal protuberance, did not reveal any fissures in the surface of berries that may lead to increased transpiration rates. Preveraison berry epicuticular wax platelets were defined and intricate, while postveraison and shriveled berry surfaces had large areas of amorphous waxes. Postveraison, the area of amorphous wax relative to intricate wax was not correlated with berry age or degree of shrivel. Extraction of surface waxes revealed that total wax on a surface-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 final shriveled phase was 16% of the preveraison rate on a per berry basis. Nevertheless, berry transpiration could account for an average 15 mg loss in fresh weight per berry per day. We conclude that weight loss during late ripening of Shiraz berries was not the result of cuticle disruption or high transpiration rates alone. It is hypothesized that decreased vascular flow of water into the berry combined with continued transpiration leads to the weight loss. Key words: Vitis vinifera, berry, shrivel, transpiration

Grape berries can lose weight and shrivel at advanced The cuticular membrane covers all aboveground parts of stages of maturity. Berries of Vitis vinifera L. cv. Shiraz are terrestrial plants, including their fruits. This membrane con- unusual in that the weight loss can occur well before the sists of a polymer matrix (cutin), polysaccharides, and sol- berries are suitable for harvesting, even under adequate ir- vent-soluble lipids (cuticular waxes) (Holloway 1982). The rigation (McCarthy 1999). This weight loss can begin 80 to cuticle controls water movement between the epidermal 115 days after flowering, depending on season, and can cells and the ambient atmosphere (Riederer and Schreiber reach up to 30% of maximum weight prior to harvesting. 2001). When present, stomata are the preferred sites of wa- Nearly all of the weight loss (90%) is in the form of water ter loss. In grape berries, however, their frequency is even vapor, while loss in dry weight could be accounted for by lower than that of leaves of crassulacean acid metabolism berry respiration (Rogiers et al. 2000). Impeded vascular plants (Blanke and Leyhe 1987), and it is likely that the cu- flow from the vine into the berry during the later growth ticle is important in controlling water loss. The structural stage has been hypothesized to play a role in shriveling at arrangement of the wax, together with its chemistry, con- suboptimal Brix (McCarthy and Coombe 1999). Unusually trols the water movement from berries (Chambers and high berry transpiration rates may be another physiological Possingham 1963, Possingham et al. 1967). The structure of cause for this condition, leading to excessive water loss epicuticular waxes changes with fruit age. Although nor- (Düring et al. 1987, Lang and Thorpe 1989). If influx of wa- mally crystalline, the wax material is rather soft and can be ter to the berry through the pedicel cannot compensate for altered or removed by the impact of rain, abrasion from water loss through the cuticle, then the shriveling symp- wind-blown particles, or contact with other berries, leaves, toms often observed in Shiraz may result. or other adjacent objects. On some leaf surfaces investigated, the crystals have appeared to degrade slowly over time, fusing into amorphous masses and eventually into a continuous layer on top of the normal, uninterrupted wax 1Cooperative Research Centre for Viticulture, PO Box 154, Glen Osmond, SA 5064, Australia; 2National Wine and Grape Industry Centre, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, layer (Reicosky and Hanover 1976, Grill et al. 1987). 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; Reynhardt and Riederer (1991) proposed a model of the and 5Washington State University, Irrigated Agriculture Research and Extension Center, Prosser, WA 99350 USA. physical structure of the epicuticular wax layer, describing *Corresponding author [Fax: +61 2 6933 2107, email [email protected]] it as a matrix composed of highly ordered crystalline and Acknowledgments: This research was supported by the Commonwealth Cooperative Research Centre disordered amorphous regions. Reduced diffusional dynam- Program and conducted through the CRC for Viticulture with support from Australia’s grapegrowers and winemakers through their investment body the Grape and Wine Research and Development Corporation, ics in the crystalline regions of the wax barrier make them with matching funds from the federal government. We thank Paul Kriedemann for helpful discussions during the course of this study. Technical assistance by Robert Lamont is also gratefully acknowledged. practically impermeable. Thus, transport across the wax bar- Thanks to the Charles Sturt Winery for permitting fruit sampling and to the vineyard staff for vine rier may be expected to occur only through the amorphous re- maintenance. Manuscript submitted October 2003; revised December 2003, February 2004 gions. The physical state of the wax layer, more than its chemi- Copyright © 2004 by the American Society for Enology and Viticulture. All rights reserved. cal composition, may determine the transport properties of the 121

Am. J. Enol. Vitic. 55:2 (2004) 122 – Rogiers et al. cuticle. It is therefore important to investigate the relative measured with callipers, and surface area was estimated distribution of crystalline and amorphous regions on the according to the modified equation for a sphere: surface Shiraz berry and to determine if a potential transition from area = π(width)(height). Pedicels were dipped in paraffin crystalline to amorphous wax is related to the shrivel phe- wax to prevent water loss from this exposed surface. Berries nomenon. Moreover, cuticle disruption in fruits can be due were suspended in an incubator at 25°C with silica gel to to microbial pathogens or sun exposure and extreme tem- maintain a constant 23% relative humidity without air turbu- peratures. Cracks in the cuticle of expanding berries have lence. Berries were weighed twice daily over three days, and also been reported for some varieties (Percival et al. 1993, transpiration rates were calculated from the slope of the lin- Comménil et al. 1997). The presence of these cracks would ear regression of weight loss over time (the r2 of each slope provide open wounds, and once damaged, the berry may be was ≥0.994 ), taking into account an average daily respira- more susceptible to high rates of water loss. tion rate of 1.9 mg CO2 per berry (Niimi and Torikata 1979). The objectives of the present work were to examine the surface of the Shiraz berry during late ripening and shrivel- Results ing for evidence of cuticular disruption and to correlate Berry weight. In the 1999 to 2000 season, gain in berry properties of the waxy cuticle with transpiration of indi- fresh weight was biphasal with a central transition point vidual berries during development. (Staudt et al. 1986, Blanke 1992) one week before veraison (67 days after flowering, DAF) (Figure 1A). Mean berry Materials and Methods fresh weight reached a maximum at 95 DAF (1.5 g), and then declined by 20% to 1.2 g at 115 DAF, giving an average Berry weight. Five bunches of berries were harvested at weight loss of 15 mg per berry per day. In the 2001 to 2002 random from field-grown Shiraz vines twice weekly from flowering to harvest over the 1999 to 2000 and the 2001 to 2002 seasons. These 11-year-old, own-rooted vines were from clone PT23/N/Griffith and were located at Charles Sturt A University, Wagga Wagga, NSW, Australia. Fifty-berry sam- ples were taken at random from each bunch and weighed. A Epicuticular wax extraction. Fifty berries from three bunches of each sampling date of the 1999 to 2000 season were weighed and briefly rinsed in water to remove dirt and dust. They were dried on filter paper and then dipped in chloroform three times in separate vials (30 sec, 10 sec, 1 sec). Chloroform extracts were combined in tarred vials and allowed to evaporate at 22°C under nitrogen flux overnight and then dried at 40°C for 5 to 7 days until constant weight. Water-soluble exudates were partitioned from the waxes by dissolving in water (5 mL), then chloroform (5 mL). The water layer was removed with a pipette and the chloroform layer was dried as described above. B Scanning electron microscopy. Shiraz berries were B sampled during the 1999 to 2000 season at random from the field vines described above, taking care not to make contact with the surface of the berry. Vitis vinifera L. cvs. Cabernet Sauvignon and Chardonnay berries were also sampled from the same vineyard. Some berries were plunged into liquid nitrogen, and 1-cm2 skin sections were freeze-dried, attached to aluminum stubs, and sputter-coated with gold at 0.05 mbar and 25 mA for 3 min prior to scanning electron microscopy (SEM). In a second method, a 1-cm2 slice was cut from each berry, attached to a flat brass stub, quick-fro- zen at approximately -170°C on the cold stage of a JEOL 6400 SEM (JEOL Australasia Pty Ltd, Sydney, Australia), gold-coated, and then observed. Transpiration. Berries of five developmental stages (six Figure 1 (A) Berry fresh weight (n = 5, 50 berries per sample) and (B) replicates) from preveraison to shriveled were harvested at berry epicuticular wax amounts (n = 3, 50 berries per sample) during development and ripening. Inset refers to mass of chloroform extract random from the field vines described above during the prior to partitioning with water to remove berry exudates (n = 3, 50 2001 to 2002 season. Height and width of each berry were berries per sample). Bars indicate standard error of the mean.

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season, veraison again occurred at 67 DAF and the berry weight maximum occurred at 106 DAF (1.6 g). Berries were harvested at 133 DAF when they weighed 1.2 g. Weight loss was 25%, with an average weight loss of 15 mg per berry per day as in the 1999 to 2000 season. Epicuticular wax. Mass of the primary chloroform extract per unit berry surface area prior to water extraction was 1.4 ± 0.1 µg mm-2 and did not change through development (Figure 1B). After removal of water-soluble berry- surface exudates from the chloroform extract, mass on a surface-area basis ranged between 1.1 and 1.2 µg mm-2 from 35 to 50 DAF, then declined by 90 DAF (berry weight maximum) to 17% of peak values (0.2 to 0.4 µg wax mm-2) (Figure 1B) and remained low through the weight- loss phase until berries were harvested at 110 DAF. In contrast, the water-soluble exudates increased from approximately 18% of the total surface exudates at 35 to 50 DAF to 83% of exudates by 90 DAF. Morphology of the grape berry. The morphology of the grape berry changed during fruit ontogeny and ripening. At anthesis the ovary was covered in cuticular ridges (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 than five per berry) were also visible (Figure 2D). Stomates were visible on the flower cap prior to bloom, on the ovary, and on the preveraison berry. These stomates were hidden once the epicuticular waxes covered the berry. One or two stomates could be seen on postveraison berries, but they were mostly filled by the wax (data not shown). At 40 DAF the waxes formed well-defined plates with fringed edges and were so dense that the cuticle membrane underneath was no longer visible (Figure 2E, F, G). In the postveraison berry, the cuticle and epicuticular wax was approximately 2-µm thick (Fig- ure 2H). The defined vertical platelets also featured in the postveraison and post-weight maximum berries (Fig- ure 3A, B, C) and were similar to those seen on Cabernet Sauvignon (Figure 3D) and Chardonnay (data not shown). From veraison onward, large areas of amorphous wax were also observed in all three varieties (Figure 3E). The top ends of the vertical platelets appeared to spread into umbrella-like horizontal plates that condensed to form a smooth crust (Figure 3F, G). These smooth areas covered from 10 to 80% of the berry and the area was not dependent on the age of the postveraison berry or the degree of shrivel. However, sun-exposed berries had larger areas of the amorphous wax than shaded berries. The wax was also amorphous in those areas where ber- Figure 2 Scanning electron micrographs of grape berry surfaces at various ries were in contact with other berries. developmental stages. (A) Cuticular ridges on the surface of Shiraz ovary at anthesis. (B) Surface of Shiraz preveraison berry at 20 DAF. (C) Close- Cavities or fissures were not visible in plump, slightly up of (B) showing cuticular ridges and rudimentary wax granules. (D) Stomate shriveled, or moderately shriveled Shiraz berries. How- on berry surface at 20 DAF. Note the lack of wax granules near the opening. ever, there were tiny cracks along some of the stomatal (E–H) Typical berry surface at 40 to 120 DAF featuring upright, intricate protuberances in several of the postveraison berries. scalelike wax platelets. (H) Transverse section of the dermal and cuticle layers of a postveraison berry; cuticle, including epicuticular wax layer, ~2 Moreover, very tightly shriveled berries also had cavi- µm thick. Bar in A, F, H = 10 µm; bar in B, D = 20 µm.; bar in C, G = 5 µm; bar ties in the surface (Figure 3H). in E = 100 µm.

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

Discussion The epicuticular wax development on Shiraz berries in this study was similar to that described earlier for other varieties, for example, cvs. Thompson Seedless (Rosen- quist and Morrison 1988) and Palomino fino (Casado and Heredia 2001). Cuticular ridges were present at flowering, and these spread out and fused as the berry expanded. Rosenquist and Morrison (1988) suggested that the cuticular ridges function as a storage form of cuticular material, which spreads as the berry expands. The first epicuticular wax appeared as small grains and then formed into intricate crystalline platelets 20 to 40 days after flowering. This was followed by the development of an amorphous wax layer on top of the existing platelets postveraison. The proportion of the berry surface that was covered with amorphous wax was larger on sun-exposed berries than on shaded berries, perhaps related to the substantially higher temperature experienced by the exposed berries. Such berries often reach 15°C above ambient temperature, whereas shaded berries are generally close to ambient temperature (Smart and Sinclair 1976, Spayd et al. 2002). The surface wax of Valencia orange fruit changed from upright plates to amorphous during development, a change that occurred earlier for exposed fruit (El Otmani et al. 1989). There were extensive areas of amorphous wax on postveraison berries in our study. However, shriveled berries did not have more extensive amorphous wax than plump postveraison berries, sug- gesting that shriveling in Shiraz berries is not the result of amorphous cuticular wax development. Fissures on skins of berries may lead to increased transpiration 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 fissures along the surfaces of postveraison, mildly shriveled, or moderately shriveled Shiraz berries. Cabernet franc berries are relatively small, and like Shiraz berries in this study, these did not show any fissures traversing the cu- Figure 3 Scanning electron micrographs of Shiraz grape berry surfaces. ticle (Percival et al. 1993). Cuticles of varieties with larger (A) Moderately shriveled berry at 100 DAF. (B) Close-up of (A) showing berries may be more susceptible to fissures because there intricate wax platelets. (C) Intricate wax platelets of severely shriveled is lack of adequate mechanical support (Percival et al. berries at 120 DAF; note fungal hyphae. (D) Wax platelets of Cabernet Sauvignon postveraison berry, similar to that of Shiraz. (E) Amorphous 1993). In some varieties small cracks can develop at the wax formation seen on portions of Shiraz, Chardonnay, and Cabernet base of stomatal protuberances during veraison (Blanke Sauvignon berries 60 to 120 DAF. These waxes can cover up to 80% of et al. 1999). We also observed these cracks on some post- the berry. (F, G) Close-up of (E) showing formation of amorphous waxes veraison berry stomata, but, as this phenomenon occurs on top of platelets. (H) Cavities in the surface of tightly shriveled and decaying berries (not present in mildly or moderately shriveled berries). in many varieties, it is unlikely that it would lead to the Bar in A, E, H = 100 µm; bar in B, C, D = 10 µm; bar in F, G = 5 µm. shriveling that is unique to Shiraz. The cavities in the se-

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Table 1 Transpiration rates of five developmental stages of Shiraz berries sampled on As indicated above, in other varieties the same day. Loss in fresh weight of detached berries monitored over three days at there appears to be no consistent corre- 25°C and 23% relative humidity (VPD = 2.45 x 10-3 MPa). Transpiration rate calculated lation between berry development and by subtracting a daily respiratory loss of 1.9 mg CO /berry (Niimi and Torikata 1979) 2 wax thickness or quantity per surface as carbon from fresh weight loss. Values are means ± standard errors. area. For example, the wax of Delaware Berry maturity Soluble solidsa Transpiration rate Transpiration rate grapes increased during the early pre- (Brix) (µmol m-2 s-1) (µmol berry-1 s-1) veraison stage (until 30 DAF) and then Green (preveraison) —b 129 ± 3 0.062 ± 0.002 remained constant thereafter (Yamamura Pink (50%) 22.6 ± 0.7 62 ± 2 0.030 ± 0.001 and Naito 1983). In Thompson Seedless Pink (100%) 22.6 ± 0.7 47 ± 2 0.023 ± 0.001 grapes, however, no change in the total Blue (postveraison) 31.2 ± 0.9 24 ± 1 0.0117 ± 0.0006 -2 Shriveled 44.0 ± 0.8 21 ± 1 0.0098 ± 0.0006 wax (1 µg mm ) during berry growth was reported (Radler 1965), and an increase aSoluble solids readings taken after three days of transpiration measurements. in the weight of wax per unit surface area bPreveraison berries did not contain adequate amounts of juice for a soluble solids assessment. occurred during development of Pinot Noir grapes (Comménil et al. 1997). Our results for Shiraz suggest that the forma- verely shriveled berries were likely a by-product of shrivel tion and deposition of epicuticular wax occurs early during as opposed to a cause of shrivel, as they were not apparent berry development and ceases before the central transition in earlier stages of shriveling. point of berry growth. These conflicting reports on amount In order to determine whether decreasing wax quantities of berry surface wax may be due to differences in methods contributed to Shiraz shrivel, chloroform was used to re- of wax extraction. Furthermore, the length of the develop- move the surface wax from the grape berries. The main com- mental period investigated and environmental factors such ponent of berry wax is a hard wax, oleanic acid, whereas as temperature, light exposure, relative humidity, and irriga- soft wax is a mixture of long-chain fatty acids, alcohols, al- tion (Rosenquist and Morrison 1989) will have an effect on dehydes, esters, and hydrocarbons (Radler and Horn 1965, trends. Yamamura and Naito 1983, Comménil et al. 1997). For ex- One might intuitively assume that the amount of epicu- ample, Yamamura and Naito (1983) found that Delaware ticular wax will affect cuticular transpiration. In this study, berry surface wax was predominantly hard wax during all however, there was no negative correlation between amount growth stages (87% at maturity). Since chloroform solubi- of wax and berry transpiration. Berry transpiration per sur- lized not only the waxes but also the other exudates on the face area decreased from preveraison to postveraison and berry surface, these exudates were removed from the chlo- then decreased even further during the shriveling phase of roform extract by washing in water. The remaining chloro- ripening. Similarly, transpiration of postveraison Müller- form layer was then dried down to constant weight. Berry Thurgau (Leyhe and Blanke, 1989) and Cabernet Sauvignon exudates consist of phenolic compounds and malic acid in (Greenspan et al. 1994, 1996) berries was less than that of immature fruit, and as the fruits mature they contain sugars, preveraison berries. The composition of the cuticle and the malic acid, potassium, and sodium (Padgett and Morrison epicuticular wax may be more important than its thickness 1990). or density for determining transpiration rates. The physical The total mass of primary chloroform extract remained structure of the wax, such as crystallinity, may also affect unchanged, on a surface area basis, throughout develop- transpiration rates (Reynhardt and Riederer 1991), but as ment. This result contrasts with the finding of Palliotti and there was no correlation between area of amorphous wax Cartechini (2001), who reported a substantial increase in and incidence of shrivel, other factors may be involved. chloroform-extracted wax (from 1 up to 5 µg mm-2) from Loss of stomatal functioning may also contribute to the postflowering to postveraison Cabernet Sauvignon berries. decreased transpiration during ripening of grape berries This contrast is starkly enhanced by removal of the water (Palliotti and Cartechini 2001). Shortly after fruit set, the sto- soluble extracts, which revealed a substantial decrease in mata were raised on a conical protuberance and appeared wax, on a surface-area basis, from veraison until the berry to be functional. An “aureole peristomatique” was first de- weight maximum (1.2 to 0.3 µg wax mm-2). This may seem scribed for the grape berry by Bessis (1972), and its protu- surprising considering that the scanning electron micros- berance was discovered and labeled “stomatal protuber- copy images indicated the postveraison development of an ance” by Blanke and Leyhe (1988). The very few stomata we amorphous layer on top of the existing wax platelets. How- observed in postveraison berries in this study had been ever, it was observed in Pinot noir grapes that, despite the transformed into nonfunctional lenticels and were primarily formation of this layer, cuticle thickness decreased as the filled with wax. The continued decrease in berry transpira- berry expanded (Comménil et al. 1997). The density of the tion after veraison is further evidence that shrivel is not wax platelets may also decrease as the berry expands, caused by fissures or cavities in the skin. Such physical which may result in an overall decrease in wax quantities damage would be expected to increase transpirational water per surface area. loss rather than to decrease it.

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Transpiration rates of shriveled berries were equal to the Literature Cited rates of weight loss observed in field berries (on average -1 -1 15 mg water vapor berry day ). However, since transpira- Bessis, R. 1972. Etude de l’évolution des stomates et des tissues tion rate decreased on both a surface-area and whole-berry péristomatiques du fruit de la vigne. C.R. Acad. Sci. Paris, t. 274, basis during ripening, it is unlikely that this alone would be Sér. D:2158-2161. the cause for shrivel. In a growing postveraison berry Blanke, M.M., and A. Leyhe. 1987. Stomatal activity of the grape (prior to the weight maximum), net water flow into the berry berry cv. Riesling, Müller-Thurgau and Ehrenfelser. J. Plant Physiol. must exceed net water loss. In a shriveling berry, the rate of 127:451-460. water loss through transpiration was slightly less than that Blanke, M.M., and A. Leyhe. 1988. Stomatal and cuticular transpi- of the growing postveraison berry, indicating that net water ration of the cap and berry of grape. J. Plant Physiol. 132:250- flow into this shriveling berry must have been less than 253. that of the growing postveraison berry. If transpiration Blanke, M.M. 1992. Lag phase during grape berry growth: Fact or rates had suddenly increased during shriveling, then tran- artefact? Wein-Wiss. 47:147-150. spiration alone could have accounted for the transforma- Blanke, M.M., R.J. Pring, and E.A. Baker. 1999. Structure and el- tion of a plump berry into a shriveled one. While Coombe emental composition of grape berry stomata. J. Plant Physiol. and McCarthy (2000) have hypothesized that xylem flow 154:477-481. into the berry decreases at veraison and that phloem flow is Casado, C.G., and A. Heredia. 2001. Ultrastructure of the cuticle discontinued at the weight maximum, earlier studies using during growth of the grape berry (Vitis vinifera). Physiol. Plant. element accumulation patterns indicated that xylem flow 111:220-224. into the berry is not necessarily completely impeded after Chambers, T.C., and J.V. Possingham. 1963. Studies of the fine struc- veraison (Ollat and Gaudillère 1996, Rogiers et al. 2000). ture of the wax layer of Sultana grapes. Aust. J. Biol. Sci. 16:818- Tracer dye studies pointed to a xylem disruption between 825. the brush region of the berry and the rest of the berry Comménil, P., L. Brunet, and J.C. Audran. 1997. The development (Rogiers et al. 2001). However, this disruption does not of the grape berry cuticle in relation to bunch rot disease. J. Exp. necessarily indicate a complete cessation of xylem ex- Bot. 48:1599-1607. change between the vine and the berry since water could Coombe, B.G., and M.G. McCarthy. 2000. Dynamics of grape berry potentially flow into the berry through the xylem up to the growth and physiology of ripening. Aust. J. Grape Wine Res. brush and then continue along symplastic routes (be- 6:131-135. tween adjacent cells through plasmodesmata) from the Düring, H., A. Lang, and F. Oggionni. 1987. Patterns of water flow brush into the pulp and skin regions. Although not thought in Riesling berries in relation to developmental changes in their to be important after veraison (Greenspan et al. 1994), xylem morphology. Vitis 26:123-131. backflow has been reported in grapes (Lang and Thorpe El Otmani, M., M.L. Arpaia, C.W. Coggins Jr., J.E. Pehrson Jr., and 1989) and may play a role in shrivel. Further elucidation of N.V. O’Connell. 1989. Developmental changes in ‘Valencia’ orange this phenomenon, together with data on rates of phloem fruit epicuticular wax in relation to fruit position on the tree. Sci. flow into the berry, is required to better understand the Hortic. 41:69-81. physiological cause of shriveling in Shiraz berries. Greenspan, M.D., K.A. Shackel, and M.A. Matthews. 1994. De- velopmental changes in the diurnal water budget of the grape berry Conclusion exposed to water deficits. Plant Cell Environ. 17:811-820. Greenspan, M.D., H.R. Schultz, and M.A. Matthews. 1996. Field The objective of this study was to examine the role of evaluation of water transport in grape berries during water defi- berry transpiration in Shiraz berry shrivel. Epicuticular wax cits. Physiol. Plant. 97:55-62. amounts on a surface area basis declined after veraison, Grill, D., H. Pfeifhofer, G. Halbwachs, and H. Waltinger. 1987. In- but did not decline further after the berry weight maximum. vestigations on epicuticular waxes of differentially damaged spruce The epicuticular wax platelets were intricate or amorphous, needles. Eur. J. Forest Pathol. 17:246-255. with no correlation between the area of amorphous wax Holloway, P.J. 1982. The chemical constitution of the plant cuticle. and incidence of shrivel. There were no fissures in the In The Plant Cuticle. D.F. Cutler et al. (Eds.), pp. 45-86. Academic surface of the cuticle that might lead to increased transpi- Press, London. ration rates. Small cracks were visible along the stomatal Lang, A., and M.R. Thorpe. 1989. Xylem, phloem and transpira- protuberance, but that was not likely associated with tion flows in a grape: Application of a technique for measuring shrivel as the cracks were present in preweight maximum the volume of attached fruits to high resolution using Archimedes’ berries and are fairly common among other grape varieties. principle. J. Exp. Bot. 40:1069-1078. As fruits ripened and subsequently shriveled, there was a Leyhe, A., and M.M. Blanke. 1989. Carbon economy of the grape decline in transpiration on both a whole-berry and surface- inflorescence. CO2 exchange of the grape inflorescence. Vitic. Enol. area basis. Shriveling in Shiraz was not caused by a sudden Sci. 44:147-150. spike in transpiration rates. Rather, we hypothesise that a McCarthy, M.G. 1999. Weight loss from ripening berries of Shiraz decrease in net vascular flow into the berry together with grapevines (Vitis vinifera L. cv. Shiraz). Aust. J. Grape Wine Res. continued transpiration leads to the weight loss. 5:10-16.

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