Fruit ripening in Vitis vinifera L.: possible relation of veraison to turgor and berry softening_

278 Turgor and veraison Australian Journal of Grape and Wine Research 15, 278 283, 2009 Fruit ripening in Vitis vinifera L.: possible relation of veraison to turgor and berry softening_060 278..283 M.A. MATTHEWS 1, T.R. THOMAS 1 and K.A. SHACKEL 2 1 Department of Viticulture and Enology, University of California, Davis, CA 95616, USA 2 Department of Plant Sciences, University of California, Davis, CA 95616, USA Corresponding author: Professor Mark A. Matthews, fax 01 530 752 0382, email mamatthews@ucdavis.edu Abstract Background and Aims: Vitis vinifera L. berries exhibit dynamic changes in water relations during development, but the possible connections between water relations, particularly cell turgor pressure (P), and ripening have received little attention, and the water relations have been studied by mostly indirect methods. Methods and Results: The cell pressure probe was utilized to examine directly the in situ P of cells in the mesocarp. Mesocarp cell P demonstrated a consistent pattern of a high value early in development, followed by a decrease to less than 1.0 bar that was maintained during ripening. Sugar accumulation did not increase significantly until P had declined to less than 1.0 bar. Fruit elasticity was used to evaluate fruit firmness during development. Fruit elasticity changed dynamically and synchronously with P. When growth was prevented with plastic boxes, the decline in cell P was delayed over 14 days, and the onset of skin colour and sugar accumulation was similarly delayed. Conclusions: The results show that when the decrease in P was delayed, the onset of ripening was delayed, and, therefore, are consistent with a central role of P in the onset of ripening. Significance of the Study: This study showed that boxing preveraison berries similarly delayed P decrease and colour increase in Cabernet Sauvignon berries. Thus, this system may be useful to study the relationship between P and gene expression in developing berries. Abbreviations DAA days after anthesis; E elasticity; P turgor pressure Keywords: cell pressure probe, elasticity, grape, sugars, water deficit Introduction The onset of ripening in grape berries is characterized at the physiological level by fruit softening, resumption of growth, sugar accumulation and colour development in pigmented varieties. The onset of ripening has been frequently identified by increasing fruit softness, measured as deformation with a skin fold caliper (e.g. Coombe and Bishop 1980). Although increased deformation is attributed to an increase in cell wall elasticity (Coombe and Bishop 1980, Coombe 1992), a reduction in cell turgor pressure (P) could have the same effect. We recently showed that mesocarp P decreases approximately at veraison (Matthews and Shackel 2005) and becomes insensitive to water deficits after veraison (Thomas et al. 2006). The low P in mesocarp cells was observed in grape berries when there is fruit softening (Thomas et al. 2008) and rapid phloem influx (Greenspan et al. 1994, 1996). Based on those and other correlations, Thomas et al. (2008) suggested that low P may act as a signal for the onset of ripening. However, the relation of lower P with resumed growth is not understood, and no connection of P with colour development has been established. In this study, we investigate the relationships among these physiological aspects of ripening by perturbing the normal decline in P before veraison using plastic jackets to inhibit growth. Materials and methods Plant material and boxed berry treatments Experiments were conducted on 2-year-old, greenhousegrown Vitis vinifera L. cvs. Pinot Noir and Cabernet Sauvignon (30/20 3 C; 40/70% 10% RH; natural light with a daily maximum of 1200 mmol photons/m 2 /s 1 PAR) and on some field-grown Zinfandel at the University of California, Davis, CA, USA (38 32 N latitude and 121 46 W longitude, elevation 18 m above sea level). Greenhouse vines were pruned to two shoots with one or two clusters per shoot, and watered daily with 280 ml of nutrient solution (0.11 g/l of GrowMore (Gardena, CA, USA 90248) 4-18-38, boron removed). For some berries, doi: 10.1111/j.1755-0238.2009.00060.x

Matthews et al. Turgor and veraison 279 a b c d e f g Figure 1. Photographs of field experimental system in which Zinfandel berries were jacketed with plastic boxes before veraison (a d). At incipient veraison, the frequency of colour change among berries in a cluster is shown for control, solid boxed and perforated boxed treatments (e). Close-up photographs indicate that the boxes only partially stopped growth (f and g). diameter was measured repeatedly during development with hand-held calipers, returning to the same berry and same caliper orientation on each sample date. The accumulation of soluble solids was measured on small aliquots of juice from individual berries with a hand-held refractometer (Reichert A2R200, Reichert GmbH, Seefeld Germany) and reported as Brix. In order to prevent growth, some Zinfandel berries were jacketed with plastic boxes prior to veraison. Solid boxed berries were enclosed on five sides by 1-cm plastic cubes with solid walls and a parafilm seal over the pedicel area (see Figure 1). Perforated boxed berries were enclosed in similar cubic boxes, except the walls were perforated with holes (approx. 2 mm diameter). The boxes were cut from the base of disposable, plastic spectrophotometer cuvettes; ventilation holes were created with a hot soldering iron. The boxes were installed on 8 June with 12 15 berries in each treatment. Experimental berries were selected for diameters that were approximately 1 cm such that friction held the boxes in place. Assaying turgor The cell pressure probe technique was used as described previously (Shackel et al. 1987) to measure the P of individual cells in the mesocarp of grape berries at depths between 90 and 2000 mm from the epidermis (Matthews and Shackel 2005). All measurements were performed under laboratory conditions (diffuse fluorescent light and 25 28 C air temperature) and completed within 1 5 h of detachment from the cluster depending on the sample size. For most measurements, berries were excised at the pedicel and immediately placed into aluminium foilcoated bags that excluded light and prevented transpiration, which maintains mesocarp cell P unchanged for 2 days (Thomas et al. 2006). Berry rheological measurements Berry softening was measured as described previously (Coombe and Bishop 1980) by placing a Harpendon skinfold caliper (British Indicators Ltd, St Albans, UK) along the equator of the berry, recording the initial diameter (d i), releasing the calipers to apply the force of the spring to the berry and recording a second reading (d f): D( ) = ( d d ) mm i f The response of the berry to an applied load was expressed by an alternative method (Thomas et al. 2008) in which elasticity (E) was calculated from the Hertz equation: ( ) ( ) E( MPa) = 3F 1 v 2RD 2 3 12 where F is force (N), n is Poisson s ratio (dimensionless, assumed = 0.5) and R is the undeformed sphere radius (mm). The Hertz equation corrects for the geometrical effects of both D and R on the area of contact between a flat plate and an elastic sphere, but neither D nor E correct for viscous effects. For these berry measurements, the initial value of D was followed by very small changes (viscous flow under an applied load), and so the initial value of D was consistently used, as in Lang and Düring (1991).

280 Turgor and veraison Australian Journal of Grape and Wine Research 15, 278 283, 2009 Figure 2. Mean P ( ) and Brix (D) of berries sampled from greenhouse-grown Cabernet Sauvignon vines at various days after anthesis. Turgor is the mean of up to 6 10 cells in two berries, and Brix is the soluble solid content of those berries. Error bars represent the 95% confidence interval. Arbitrary designations of stages I, II, III and veraison are indicated. Results In greenhouse-grown Cabernet Sauvignon berries, P had a consistent mean value of ~1.6 bars during early stage I (Figure 2). At 36 days after anthesis (DAA), P began to increase, and reached a maximum of 3.0 bars at 48 DAA. P then declined during stage II to about 0.5 bar by 60 DAA, veraison, which was maintained throughout ripening. When P in the berry mesocarp declined from 3.0 to 1.6 bars (between 49 and 56 DAA), there was a negligible increase in soluble solids (Figure 2). During a further drop in P to less than 1.0 bar, soluble solids increased 3.6 Brix in 3 days (between 56 and 59 DAA) and continued increasing thereafter. Thus, rapid sugar accumulation did not begin until P was approximately 1.0 bar or less. In greenhouse-grown Pinot Noir, the developmental pattern of mesocarp cell P (Figure 3) was similar to that in Cabernet Sauvignon, although there was no clear increase in P during stage I. Nevertheless, mean cell P was 2.6 bars at 32 DAA, and decreased to low values by 60 DAA, although the decrease in P started earlier in Pinot Noir than in Cabernet Sauvignon (cf. Figures 2,3). Berry elasticity (E) exhibited a similar pattern to P, increasing from about 20 to about 45 bars, and then decreasing to less than 10 bars by 60 DAA (Figure 3b). The striking decreases in P and E preceded veraison and the resumption of growth. Turgor pressure and E remained low during stage III. Throughout the developmental changes in P, there was a linear relationship between E and P (Figure 3c, r 2 = 0.81; P < 0.001). The pre-veraison P decline was present in field-grown Zinfandel as well. Cell P decreased from 1.5 to 0.3 bar between 54 and 68 DAA (controls, Figure 4). Cell P decreased in all berries as veraison was approached, but the decrease was delayed when individual Zinfandel berries were boxed during stage I as shown in Figure 1a d. Turgor in solid boxed berries was maintained 0.8 bar higher than in controls at 68 DAA, and 0.4 bar higher than in controls at 82 DAA (Figure 4). Turgor in Figure 3. Berry diameter (a) and P and E (b) of greenhouse-grown Pinot Noir berries at various days after anthesis. Data for P are means of the same four berries (P subsampled on 8 10 cells/berry); means standard deviation. Linear regression of E with P (c) using data from (b). perforated boxed berries was similar or slightly less than in solid boxed berries (Figure 4). In both boxing treatments, a high P was present, but that was lost within 30 min of removing the boxes. For example, when the boxes were removed at 82 DAA, P quickly decreased from about 0.7 bar to about 0.2 bar (Figure 4). When plastic boxes were installed and the decline in P was delayed, veraison was also delayed (Figure 1e). In controls, P declined from 1.5 bars to less than 1.0 bar at 59 DAA, whereas in solid boxed berries P was still at 1.16 bars at 68 DAA. The boxes inhibited berry growth and resulted in cube-shaped berries (Figure 1f,g). In addition to

Matthews et al. Turgor and veraison 281 restricted growth and delayed P loss, solid boxed berries delayed Brix increase and decreased total sugar accumulation. At 68 DAA, soluble sugars were already at 10 Brix in controls, but still at 5 Brix in solid boxed berries (Table 1). Soluble solid concentration in solid boxed berries caught up to controls by 97 DAA. Although the Brix in perforated boxed berries was similar to controls, both boxing treatments inhibited substantially the accumulation of total sugars per berry. Total sugar, estimated from the product of mean berry fresh weight and mean Brix at 98 DAA, was 0.44 g/berry in controls, but only 0.20 g/berry in solid boxed berries and 0.23 g/berry in perforated boxed berries. The transition from green to coloured was earliest in control berries, delayed for perforated boxed berries and last for solid boxed berries (Figure 1). The delay in veraison was indicated by the frequency of coloured berries in control and solid boxed treatments (Figure 1c). At 68 DAA, there were no coloured berries in solid boxed berries, two coloured berries in perforated boxed and eight coloured berries in the controls among samples of 12 berries. At 82 DAA, there were still only three coloured berries in solid boxed berries, Figure 4. Mean mesocarp cell P at various days after anthesis (DAA) for controls, solid boxed and perforated boxed Zinfandel berries before boxes were removed and after boxes were removed (box off). Data are means standard deviation of four to eight cells per berry in two or three berries, except for solid boxed and perforated boxed on 96 DAA where data are reported from a single berry. seven coloured berries in perforated boxed berries and almost all coloured berries in controls. By 97 DAA, all berries were coloured. In both solid boxed and perforated boxed berries at 97 DAA, mean berry fresh weight and diameter were reduced approximately 55 and 30%, respectively (Table 1). Discussion Although the control and coordination of softening, sugar accumulation and resumption of growth at the onset of ripening await clarification, a central role of P in each has been hypothesized. Direct measurements of mesocarp cell P in field-grown Zinfandel showed that P is high before veraison and low after veraison, and that the decrease in P precedes rapid sugar accumulation. The solid boxed treatment that prevented stage III growth also delayed the decrease in P and the onset of ripening as indicated by sugar accumulation and colour development. These observations are consistent with the suggestion that the precipitous decline in mesocarp P plays a role in the onset of grape berry ripening (Thomas et al. 2008). Coombe and McCarthy (2000) tied the onset of sugar accumulation to the resumption of growth, which was thought to stretch peripheral xylem conduits to failure and, by implication, to water deficit reflected in lower P. Our results confirm a connection between growth and veraison. However, the growth restriction of the two boxing treatments did not alter the water transport properties of berry xylem (Bondada et al. 2005), and the system of xylem conduits remains functional past veraison (Keller et al. 2006, Chatelet et al. 2008). Therefore, an alternate explanation for the connection must be sought. Although there have been a few studies in which physical growth restraints were employed, there appear to be no similar studies where growth was restricted in sugar-accumulating sinks. Jacketing soy bean pods delayed seed maturation (seed dry down) and decreased the rate of leaf senescence (Crafts-Brandner and Egli 1987). In growth restriction experiments with tomato (Kawabata et al. 2002) and cucumber (Kawabata and Sakiyama 1998), concentrations of sugars increased from about 2 to 3% fresh weight. In similar treatments with turnip (Kawabata et al. 1992), sugar accumulation was unaffected. The concentration of sugars was very low in these sinks compared to grape berries. Table 1. Fresh weight, Brix and diameter of Zinfandel berries that were exposed to different growth constraint treatments. Treatment 68 DAA 97 DAA Brix Fresh weight (g) Brix Fresh weight (g) Diameter (mm) Control 10.2 (1.5) 2.044 (0.43) 16.1 (4.2) 2.71 (0.69) 16.3 (1.5) Solid boxed 5.3 (0.3) 1.058 (0.11) 17.5 (3.7) 1.14 (0.15) 11.4 (0.66) Perforated boxed 10.2 (2.6) 1.172 (0.14) 20.2 (3.5) 1.16 (0.15) 11.5 (0.38) Solid boxed berries were enclosed on five sides by 1-cm plastic cubes with solid walls and a parafilm seal over the pedicel area. Perforated boxed berries were enclosed similarly in similar cubic boxes, except the walls were perforated with holes (approx. 2 mm diameter). Boxes were installed on 8 June, and anthesis was approximately 1 May. Data are means standard deviation of 12 or 13 berry samples.

282 Turgor and veraison Australian Journal of Grape and Wine Research 15, 278 283, 2009 The boxed treatments must have inhibited fruit transpiration, as well as fruit expansion. For berries that were solid boxed, the decrease in P was delayed over 14 days, and the increase in soluble solutes and skin colour was similarly delayed. When the boxes were ventilated with holes (perforated boxed), the decline in P and onset of colour was less delayed, and sugar accumulation was not significantly delayed. The intermediate effects of the ventilated boxes suggest a role of transpiration that requires water transport through the apoplast. The relationship between fruit transpiration and phloem influx has remained elusive. Dreier et al. (2000) argued that phloem influx to the berry is transpiration dependent, but did so without the benefit of treatments to perturb the relationship. In experiments to manipulate berry transpiration, results of Rebucci et al. (1997) gave mixed indications of effects on sugar uptake, but the positive results were confounded by changes in berry temperature that may have affected both transpiration and phloem metabolism. In tomato, Johnson et al. (1992) argued from measurements of fruit and stem water potentials that the water potential gradient between fruits and stems is dependent mostly on the water potentials of the source organ rather than the fruit, and, therefore, the effect of transpiration from the fruits on phloem transport is small. In an imaginative series of experiments, sugar uptake in tomato was affected by changes in fruit transpiration only when tomato fruit growth was also physically restricted (Kawabata et al. 2005). The results imply that only under artificial conditions would transpiration of fruits contribute to carbohydrate transport; it does not serve as a limiting step of carbohydrate transport to tomato fruits under normal circumstances. In the post-veraison berry, most water influx is via the phloem, similar to the tomato. The intermediate results in the perforated boxed treatment and the positive effect of transpiration on sugar influx in growthrestrained tomato indicate that the inhibition of sugar and colour accumulation in the solid boxed treatment was partially because of inhibited berry transpiration. Coombe and Bishop (1980) carefully analysed berry size and deformation, and concluded that fruit softening (as determined by deformation in a skin fold caliper) occurs rapidly and is the earliest evidence of the onset of ripening. However, if softening is measured as amount of deformation relative to the size of the berry and the force applied, described here as E, softening begins earlier than their interpretation of veraison, and the sudden softening is not apparent (Thomas et al. 2008). Indeed, P and E both begin decreasing approximately 10 days before the transition from stage II to stage III (present study, Thomas et al. 2008). The high and positive correlation between P and E in greenhouse-grown Pinot Noir in this study is similar to the correlation found in greenhouse-grown Cabernet Sauvignon and Chardonnay (Thomas et al. 2008) and in field-grown Chardonnay (Wada et al. 2008). These correlations are consistent with the suggestion that most fruit softening in grape is attributable to P loss (Thomas et al. 2008), although most authors attribute fruit softening to cell wall disassembly (Vicente et al. 2007). Whether there is a mechanistic relation between P and the other physiological aspects of ripening is not clear. However, the circumstantial and temporal connection of P to all of these in the present and other recent studies (Thomas et al. 2008, Wada et al. 2008) invites speculation of its role in the suite of gene expression and metabolic changes that occur at veraison. Turgor probably decreases from a maximum of 2 3 bars to a stable low value of about 0.5 bar in wine grapes in general, because this has been observed in all varieties examined Chardonnay, Cabernet Sauvignon, Pinot Noir and Zinfandel. The transition from green to red skin in coloured varieties is, like softening and sugar accumulation, indicative of the onset of ripening and may also depend on the decline in P, although less directly. Both the decline in P and the development of colour were delayed in boxed berries, and the delay was less for both processes in ventilated boxes than in solid-walled boxes. The onset of anthocyanin biosynthesis is promoted by, if not dependent upon, the influx of sugars (Gollop et al. 2002, Castellarin et al. 2007a), which is thought to be dependent on low sink P (Lang and Düring 1991, Johnson et al. 1992, Patrick 1997). Furthermore, berry skin colour is enhanced by exogenous application of the stress hormone abscisic acid (ABA) (Jeong et al. 2004). Decreased P leading up to veraison should contribute to the increased concentration of ABA in berries at or near veraison (Coombe and Hale 1973) by promoting either de novo synthesis (Castellarin et al. 2007b) or ABA import (Huang et al. 2001). Although there is some evidence for P-regulated gene expression (e.g. Jones and Mullet 1995), there is surprisingly little direct investigation of P in most studies of fruit ripening (Neill and Burnett 1999). 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