Effect of wounding on cell wall hydrolase activity in tomato fruit

Postharvest Biology and Technology 40 (2006) 250–255

Effect of wounding on cell wall hydrolase activity in tomato fruit Thanh Tu Chung, Gillian West, Gregory A. Tucker ∗ The University of Nottingham, School of Biosciences, Division of Nutritional Sciences, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK Received 16 September 2005; accepted 12 February 2006

Abstract Changes in the activity of the cell wall hydrolases – polygalacturonase (EC 3.2.1.15), pectinesterase (EC 3.2.1.11) and ␤-galactosidase (EC 3.2.1.23) – have been investigated following wounding of tomato fruit pericarp tissue (Lycopersicon esculentum cv. Ailsa Craig). In ripening fruit wounding appears to arrest the further synthesis of polygalacturonase. ␤-Galactosidase synthesis may also have been arrested in ripening fruit. The level of pectinesterase declined over the first 24 h following harvest, and since this was apparent in both wounded and unwounded tissue may be related to a harvest, rather than a wounding effect. There was a recovery of activity in intact fruit by 48 h after harvest but this seems to be impaired in wounded tissue. In the case of pectinesterase, this observation was extended to examine the changes in isoform profile and it appeared that the decline of this enzyme may be associated with the reduction of one specific isoform — PE2. In contrast to ripening fruit, wounding of fruit at the fully ripe stage appears to have no significant effects on the activities of any of these three enzymes. © 2006 Elsevier B.V. All rights reserved. Keywords: Cell wall hydrolases; Pectinesterase; Polygalacturonase; ␤-Galactosidase; Wounding; Fresh-cut fruit

1. Introduction Fresh-cut fruit/vegetable salads are becoming increasingly popular for their convenience, fresh-like appearance, and the fact that they are generally free of additives. A major problem however, of fresh-cut produce is that it is subject to wounding through processing. Thus in fresh-cut produce the tissue is exposed to stress conditions which can result in the deterioration of cell wall structure and hence a reduction in shelf life. Yang and Pratt (1978) and Yu and Yang (1980) demonstrated that mechanical wounding leads to increased activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACS; EC 4.1.1.14), an enzyme which catalyses the formation of 1aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. This has been shown to occur in the albedo tissue of orange discs, and in wounded tomato fruit tissues, resulting in accelerated ethylene production. This ethylene could in turn stimulate the ripening process. Wounding is however, known to induce a much wider array of physio∗

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logical and biochemical changes within the wounded tissues involving altered expression of several genes and changes in enzyme activities (Mehta et al., 1991). These woundactivated responses are directed to healing the damaged tissue and providing defense mechanisms that prevent further damage. A review by Le´on et al. (2001) showed that following wounding, the plant activates a defense mechanism, which generally depends on the transcriptional activation of specific genes. These induced-responses may occur within a few minutes, several hours or even a few days after wounding. Softening is an important factor effecting the aesthetic quality and shelf life of fresh-cut fruit. Softening is thought to be associated with the degradation of pectic substances in the cell wall caused by the accumulation of cell wall hydrolase activities such as pectinesterase (PE), polygalacturonase (PG) and ␤-galactosidase (␤-Gal). Whilst there have been intensive studies on the role of these cell wall hydrolases during normal fruit ripening (Brummell and Harpster, 2001), very little attention has been paid to the role they may play in wounded fruit tissue. Karakurt and Huber (2003) demonstrated that PG, cellulase, and ␤-Gal activities in fresh-cut papaya all increased within 24 h of wounding and remained significantly higher than in intact fruit during storage. These

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changes were accompanied by an increase in both ACS and ACC oxidase activities raising the possibility of enhanced ethylene production stimulating general ripening processes. In this instance the papaya had been processed into relatively large pieces and it was unclear whether the responses of the tissues adjacent to, or remote from, the cut surface were similar or not. This would be an important consideration in the case of those fruit, such as tomato that during minimal processing are cut into relatively thin slices and where all the tissue could be considered as being adjacent to the cut surface. There is also the possibility that reaction to wounding may vary between fruit types and that this response may be dependent on the stage of ripening of the fruit. The purpose of this study was therefore, to investigate the effect of wounding on the activities of these cell wall hydrolases in thin slices of tomato fruit at two stages of fruit development — namely early in the ripening process one day after the first sign of colour change (breaker+1) and fully ripe.

2. Materials and methods 2.1. Plant material Tomato plants (Lycopersicon esculentum cv. Ailsa Craig) were grown in a glasshouse under controlled conditions in a cycle of 16 h light, 250 nmol−1 m s−1 photosynthesis photon flux (PPF) at 20 ◦ C, 8 h dark at 14 ◦ C. The fruit were tagged at anthesis (defined as the time of petal drop and fruit set). Fruit were harvested at two stages of ripening — breaker+1 (B+1), 1 day after the first visible sign of colour change on the fruit, ca. 40–45 days post anthesis, and orange red (OR), ca. 47–50 days post anthesis. Fruit harvested at these defined stages were divided randomly into two groups. Tissue from the first group was wounded by cutting the pericarp longitudinally (from calyx to blossom end), using a sharp knife, into 0.5 cm wide slices that were either frozen immediately in liquid nitrogen (control sample at time 0) or held at room temperature (RT) in sealed containers. The second group was kept intact, held at RT and processed as unwounded control samples. All of the samples were frozen in liquid nitrogen and stored at −70 ◦ C until required. 2.2. Enzyme extraction Twenty grams of frozen tomato pericarp tissue were ground in 4 vol (w/v) of extraction buffer consisting of 1 M NaCl and 0.05 M sodium acetate (NaOAc), pH 6.0 using a polytron homogenizer. The homogenate was adjusted to pH 7.0 with NaOH. The homogenate was kept at 4 ◦ C for 3 h and then centrifuged at 1500 × g for 30 min. The supernatant was collected and solid ammonium sulphate added to give 80% saturation (0.57 g ml−1 ). Precipitated protein was collected by centrifugation at 12,100 × g for 30 min at 4 ◦ C and dissolved in 5 ml of 0.1 M NaCl, 0.05 M NaOAc, pH 6.0.

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The resultant solution was dialysed against the same buffer overnight. This crude extract was used for all cell wall hydrolase assays. 2.3. Protein determination Protein measurement was carried out as described by Bradford (1976) with Bio–Rad protein dye reagent and bovine serum albumin (BSA, Sigma) as a standard. The protein concentration was monitored by measuring the absorbance at 630 nm and expressed as mg/g fresh weight (mg gfwt.−1 ). 2.4. Pectinesterase assay Pectinesterase activity was measured using the titration method as described by Tucker et al. (1982). Twenty microlitres of crude enzyme was assayed in 10 ml of 0.5% citrus pectin in 0.15 M NaCl, pH 8.0 at 25 ◦ C. The pH was held constant by titration with 5 mM NaOH. The reading was recorded every 30 s for 5 min. Results were calculated as ␮eq H+ min−1 . PE isoform separation was carried out using Bio–Rad heparin affinity chromatography (Cameron et al., 1998). The system consisted of an Econo system controller model ES-1, Econo pump model EP-1, Econo UV monitor model EM-1, Econo buffer selector model EV-1, six-port sample injection valve model MV-6, diverter valve model SV-3, and fraction collector model 2128. The column, with 5 ml bed volume, was equilibrated with buffer A (10 mM Tris–HCl, 10 mM NaCl, pH 7.5). Samples (2 ml) were applied in buffer A and isoforms eluted, at a flow rate of 1 ml min−1 using a linear gradient of NaCl from 10 mM (buffer A) to 600 mM (10 mM Tris–HCl, 600 mM NaCl, pH 7.5.). Fractions (75 × 2 ml) were collected and assayed for PE activity using a microtitre plate method. Twenty microlitres of each fraction was placed into wells in a 96-microtitre plate. Two hundred microlitres of assay buffer (0.5% citrus pectin, 2 mM Tris–HCl, 150 mM NaCl, 0.002% phenol red, pH 8.0) was added into each well. The plate was read on a Dynatech MR 5000 microtitre plate reader after 2 h incubation. Fractions equivalent to the two major isoform groups were pooled, concentrated by ultrafiltration using an Amicon P10 membrane, and then PE activity assayed using the titration method as described above. 2.5. Polygalacturonase assay Polygalacturonase activity was measured by the release of reducing sugars using 2-cyanoacetamide as described by Gross (1982). Twenty microlitres of crude extract was incubated in 150 ␮l assay buffer (0.5% polygalacturonic acid, 0.15 M NaCl, 0.05 M NaOAc, pH 4.0) at 37 ◦ C for 10 min. The reaction was stopped by adding 1 ml of 100 mM borate, pH 9.0, then 200 ␮l of 1% (w/v) 2-cyanoacetamide followed by incubation for 10 min at 100 ◦ C. PG activity was calcu-

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lated based on a standard curve of d-(+)-galacturonic acid and expressed as ␮mol min−1 gfwt.−1 . 2.6. β-Galactosidase assay ␤-Gal activity was assayed following the release of pnitrophenol from p-nitrophenyl ␤-d-galactopyranoside substrate. One hundred and thirty microlitres of the substrate mixture consisting of 13 mM ␤-d-galactopyranosidase, 1% BSA in 0.1 M citric acid pH 4, were equilibrated to 37 ◦ C. The reaction was started by adding 20 ␮l sample and allowed to proceed for 10 min. The reaction was terminated by adding 100 ␮l of 0.4 M Na2 CO3 . The concentration of liberated pnitrophenol was determined by measuring the absorbance at 405 nm. The activity level was calculated based on the standard curve of p-nitrophenol. One unit of activity was defined as that amount of enzyme that hydrolases the release of 1 ␮mol of p-nitrophenol min−1 at 37 ◦ C. 2.7. Statistical analysis Each experiment was repeated three times. Statistical analysis was carried out using a t-test. Data are present as the mean of triplicates and standard deviation (S.D.).

3. Results and discussion 3.1. Effect of wounding on hydrolase activities in ripening (breaker+1) fruit The protein yields from breaker+1 fruit tended to decline in both wounded and unwounded controls following harvest. However, there were no statistically significant differences observed between control and wounded fruit at any stage (data not shown). The yields of PG, PE and ␤-Gal from wounded and unwounded ripening (breaker+1) fruit are shown in Fig. 1. It can be seen that the PG yield in unwounded control fruit increased with time. This was to be expected and represents the normal increase in PG activity associated with ripening (Tucker et al., 1980). In the case of wounded tissue this increase in PG yield was reduced, compared to unwounded controls such that by 48 h following wounding the PG activity from wounded tissue was significantly (p = 0.009) less than that in the unwounded control. Similar patterns were apparent when the data were expressed as specific activities (data not shown). This would suggest that wounding has resulted in a reduction in, or even complete cessation of PG synthesis. The increase in PG activity during ripening is due to de novo synthesis (Tucker et al., 1980; Bird et al., 1988) and as such this reduction may reflect an inhibition of PG gene expression following wounding. The yield of PE fluctuated in both unwounded and wounded fruit being significantly reduced after 24 h in both cases, p = 0.018 and 0.006, respectively. This may represent a response to harvesting the fruit since the reduction over 24 h

Fig. 1. Effect of wounding on enzyme activities in ripening (breaker+1) tomato fruit pericarp. Fruit were harvested at the first sign of colour change and total enzyme extracted and assayed (time 0). Fruit were then either stored intact at room temperature (unwounded) or wounded by slicing the pericarp into thin strips which were then stored in sealed containers. Enzymes were then extracted and assayed from unwounded and wounded tissues 24 h and 48 h following harvest.

appeared to be independent of wounding. In both cases the activity appeared to recover at 48 h such that levels in the unwounded control were not statistically different to those at time 0. However, the level after 48 h in the wounded fruit had not increased as much so that there was a significant difference observed between the 48 h control and wounded

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tissues (p = 0.012). Thus in this instance wounding may have impaired the ability of the tissue to recover PE activity. In the case of ␤-Gal activity yields were found to increase slightly in control fruit but this was not statistically significant. Yields from wounded fruit tended to decline following wounding but again this was not statistically significant. However, this did result in statistically significant differences between unwounded controls and wounded tissues at both 24 h (p = 0.019) and 48 h (p = 0.027) after wounding. Thus there may be an effect similar to that seen for PG activity at this stage of ripening. The significance of this for cell wall metabolism is unclear. In this case total ␤-galactosidase was measured. It is known that in tomato fruit ␤-Gal activity can be present in at least three isoforms (Carey et al., 1995) and that only one of these is associated with ␤-galactanase activity. It is possible that levels of ␤-galactanase activity, which may be important for cell wall modification are being effected by wounding. 3.2. Effect of wounding on hydrolase activities in ripe fruit Protein yields from ripe fruit were consistent across all samples (data not shown). The yields of PG, PE and ␤-Gal from wounded and unwounded ripe fruit are shown in Fig. 2. There were no statistically significant trends in any of the enzyme activities at this stage of ripening with all three activities remaining constant in both control and wounded fruit. This suggests that wounding at this stage has no effect on either PG or ␤-Gal synthesis presumably since at this stage of ripening this is complete. Yields of PE activity were also found to be consistent across both wounded and unwounded tissues suggesting that again once ripe there is little effect on the enzyme activities. There is also no evidence of any harvest related reduction in enzyme activity at this stage of ripening. 3.3. Effect of wounding on PE isoform profiles Tomato fruit PE can be separated into at least three isoforms, PE1, PE2 and PE3 (Tucker et al., 1982). To examine the effect of wounding on the activity of these individual isoforms, extracts were prepared from tomato pericarp tissue at time 0 and 48 h after wounding. PE isoforms were then separated by heparin affinity chromatography. The resultant profiles are shown in Fig. 3. Three peaks of PE activity were detected. The first peak represents the PE2 isoform, the second peak PE3 and the third PE3. Similar profiles were found for both wounded and unwounded tissues with all three isoforms clearly differentiated. However, this profiling technique can only be used for qualitative data and is not at all quantitative. In each case therefore, those fractions equating to either PE2 or combined PE1 and PE3 were pooled, concentrated and the absolute level of PE activity in each pool measured using the titration method. These values along with the ratio of PE2 to PE1 + PE3 are shown in Table 1. These

Fig. 2. Effect of wounding on enzyme activities in fully ripe tomato fruit pericarp. Fruit were harvested at the red ripe stage and total enzyme extracted and assayed (time 0). Fruit were then either stored intact at room temperature (unwounded) or wounded by slicing the pericarp into thin strips, which were then stored in sealed containers. Enzymes were then extracted and assayed from unwounded and wounded tissues 24 and 48 h following harvest.

results suggest that the decline in PE activity following harvest and wounding arise from a selective reduction in PE2 activity since absolute levels of this isoform were found to decline whilst the combined activities of the other isoforms remained constant. In tomato, PE2 has been reported to be a fruit specific isoform (Gaffe et al., 1994) which in ripe fruit can account for as much as 80% of the total PE activity (Tucker et al.,

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Fig. 3. Pectinesterase isoform profiles from wounded and unwounded tomato fruit pericarp. Total pectinesterase was extracted from ripening tomato fruit immediately following harvest or 48 h after wounding of the pericarp tissue. Pectinesterase isoform profiles were determined for unwounded () or wounded tissue (䊉) using heparin affinity chromatography.

Table 1 Effect of wounding on tomato fruit pectinesterase isoform composition Isoform

Time (h) 0

PE1 + PE3 PE2

48

Absolute level

%

Absolute level

%

0.96 ± 0.72 1.95 ± 0.67

33 67

0.93 ± 0.76 1.05 ± 0.66

47 53

Activity is expressed as ␮eq H+ min−1 gfwt.−1 .

1982). This suggests that the response to wounding might be restricted to a ripening-specific gene for PE. This can be compared with the decline seen in the expression of PG, which is also a ripening-specific gene (Grierson and Tucker, 1983) and perhaps supports the general theory that wounding has redirected metabolism away from ripening and towards wound-repair.

4. Conclusions It is well known that ethylene synthesis is enhanced in plant tissues in response to mechanical stress such as wounding (Abeles, 1973). This is true for tomato fruit and indeed it has been shown that this is associated with the specific increase in expression of ACS-1 in wounded tomato fruit (Olsen et al., 1991). This increase in ethylene production might be expected to enhance the rate of ripening in preclimacteric fruit as indeed has been suggested by Karakurt and Huber (2003) for minimally processed papaya fruit, where there was an increased expression of enzymes involved in cell wall metabolism. However, in this paper the response of tomato fruit pericarp tissue to wounding was a clear reduction in the expression of these enzymes. It is known that plant tissues, including tomato fruit, react to wounding by the ini-

tiation of repair pathways (Mehta et al., 1991). The potential exists therefore for tissues to react differentially to wounding dependent on their proximity to the wound site. In the case of the papaya fruit these were processed into relatively large pieces with the bulk of the tissue being removed from the actual cut surface. In the experiments reported in this paper, thin slices of pericarp were used and as such all the tissue was essentially adjacent to the wound surface. This may have significance for minimally processed fruit depending on the surface to volume ratio of the fruit pieces although the effect, if any, on the texture of slices was difficult to assess. It was not possible to compare softening rates between intact fruit and slices due to increased dehydration of the latter which would also impact on texture. It is also clear from these data that the response of the pericarp tissue to wounding can be influenced by the stage of development, the response being more evident in breaker than ripe fruit. If the response does indeed involve a redirection of metabolism away from ripening and into wound repair then it would seem that this might only be initiated in tomato fruit at early stages of ripening. Wounding is a form of abiotic stress and it would thus be interesting to determine whether other stresses elicit similar responses. Barka et al. (2000) have demonstrated that hormic doses of UV-C resulted in a reduction in both softening and the activities of cell wall hydrolases in tomato fruit. Interestingly this was in intact fruit suggesting that in this instance the response to the stress may have been universal throughout the pericarp tissue.

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