- Email: [email protected]
Characterization of peroxidase-mediated chlorophyll bleaching in strawberry fruit
Phytochemistry 58 (2001) 379–387 www.elsevier.com/locate/phytochem
Characterization of peroxidase-mediated chlorophyll bleaching in strawberry fruit Gustavo A. Martı´neza,*, Pedro M. Civelloa, Alicia R. Chavesb, Marı´a C. An˜o´nb a
Instituto de Investigaciones Biotecnolo´gicas-Instituto Tecnolo´gico de Chascomu´s (IIB-INTECH), UNSAM-CONICET, Camino de Circunvalacio´n Laguna Km 6, 7130 Chascomu´s, Argentina b Centro de Investigacio´n y Desarrollo en Criotecnologı´a de Alimentos (CIDCA), Facultad de Ciencias Exactas, UNLP, CONICET, 47 y 116, 1900 La Plata, Argentina Received 2 January 2001; received in revised form 9 May 2001
Abstract Peroxidase (POX) from strawberry fruits was analyzed for its capacity to bleach chlorophyll. The partially purified enzyme preperation catalyzed the bleaching of chlorophylls and their derivatives in the presence of H2O2 and phenolic compounds. The optimal reaction conditions were 35 C, pH 5.2 and ionic strength equal to 0.2. The maximum activity was observed at 1 mM of H2O2, while higher concentrations inhibited enzyme activity. Compounds with a high affinity to the heme group, radical scavengers and reducing agents, showed an inhibitory effect. Phenolic compounds such as umbelliferone, naringenin and p-substituted monophenols acted as cofactors. Instead, other phenolic compounds tested such as caffeic acid, catechin, ellagic acid, esculin and quercetin inhibited the activity of POX on chlorophylls. Phenolic compounds extracted from strawberry fruits showed an inhibitory effect on POX-chlorophyll bleaching activity, although this effect decreased markedly during ripening. POX showed higher affinity for compounds derived from chlorophyll a than from chlorophyll b, and the enzyme preferentially degraded chlorophyll derivatives with the Mg2+ ion present and the phytol group removed. The POX-chlorophyll bleaching activity was found in all ripening stages from small green to ripe, the highest activity corresponding to large green fruits. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fragaria x ananassa; Rosaceae; Strawberry; Chlorophyll; Peroxidase; Phenolic compounds
1. Introduction Chlorophyll (Chl) degradation is a characteristic symptom of leaf senescence and fruit ripening. During senescence, chlorophyll-protein complexes are disassembled and the pigments liberated. Free chlorophylls (Chls) are highly photoactive compounds that can generate free radicals, causing damage to cell membranes (Matile et al., 1996). It has been suggested that two types of reactions are involved in Chl catabolism (Shioi et al., 1995). Type I reactions include the loss of phytol, Mg2+, and other modifications of the side chains that do not involve the breaking of the tetrapyrrolic ring present in chlorophylls. Chlorophyllase, which removes the phytol and produces chlorophyllides (Chlds), has been involved in * Corresponding author. Postal address: Camino de Circunvalacion Laguna Km 6, (7130) Chascomus, Argentina. Tel.: +54-2241424049; fax: +54-2241-424048. E-mail address: [email protected] (G.A. Martı´nez).
the first step of Chl breakdown (Trebitsh et al., 1993; Matile et al., 1996). Another enzyme involved in chlorophyll catabolism is Mg-dechelatase, which eliminates Mg2+ from Chl or Chld to produce pheophytin (Phe) or pheophorbide (Pheo), respectively (Langmeier et al., 1993; Vicentini et al., 1995). Further catabolism of Pheo involves the elimination of –COOCH3 to form pyropheophorbide, and oxidation to form hydroxypheophorbide (Shioi et al., 1991) or the formation of C132-carboxylpyropheophorbide by the action of pheophorbidase (Watanabe et al., 1999). Type II reactions involve the oxidative cleavage (bleaching) of the tetrapyrrole ring. It has been reported that the cleavage of Phe a by a pheophorbide a oxygenase is the key regulatory step in the Chl catabolism pathway (Matile and Schellemberg, 1996; Ho¨rtensteiner et al., 1998; Matile et al., 1999). However, other oxidative enzymes such as lipoxygenase (Orthoefer and Dugan, 1973), Chl oxidase, and peroxidase also appear to be involved in Chl bleaching.
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00266-7
380
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
Peroxidase (POX) is a ubiquitous enzyme located in several subcellular compartments, including chloroplasts (Johnson-Flanagan and McLachlan, 1990; Kuroda et al., 1990). Although the role of POX in Chl degradation ‘in vivo’ is controversial (Matile et al., 1999), many studies have supported its possible participation in Chl catabolism (Matile, 1980; Huff, 1982; Johnson-Flanagan and McLachlan, 1990; Johnson-Flanagan and Spencer; 1996; Maeda et al., 1998). Probably, the controversy has been caused by the existence of different chlorophyll degradation pathways (Amir-Shapira et al., 1987; Trebitsh et al., 1993) and the presence of multiple POX isoenzymes with uneven capacities to degrade chlorophylls (Kuroda et al., 1990; Adachi et al., 1996). Strawberry is classified as a non-climacteric fruit, and neither the respiration rate nor ethylene production increase as the fruit changes its color, texture and flavor (Abeles and Takeda, 1990). The chlorophyll content diminishes very quickly as the fruit ripens (Given et al., 1988; Martı´nez et al., 1994; Perkins-Veazie, 1995), and the chloroplasts disintegrate during the process (Gross, 1982; Perkins-Veazie, 1995) without conversion into chromoplasts or gerontoplasts, as occurs in tomato fruits and leaves respectively (Matile et al., 1996). These characteristics make strawberry fruit an interesting model system in which to study chlorophyll degradation during fruit ripening. In the present work, we have partially characterized the activity of strawberry peroxidase for bleaching chlorophyll, determined the influence of phenolic compounds on this activity and analyzed the change of POX bleaching activity during ripening.
2. Results and discussion We have previously reported the presence of two cationic peroxidase isoenzymes in strawberry fruit, both of which are associated with membranes and show maximum activity at the green ripening stage (Civello et al., 1995). It has been suggested that cationic peroxidases, located in the chloroplast, are involved in Chl bleaching (Kuroda et al., 1990). In order to analyze strawberry POX activity on chlorophylls, extracts from green fruits were prepared and partially purified by means of (NH4)2SO4 fractionation. The absorption spectrum of complete reaction mixtures (enzyme extract, chlorophyll, H2O2, and p-coumaric acid) showed a progressive reduction of the absorbance at 669 nm (Fig. 1). According to previous reports, this effect is due to the cleavage of the tetrapyrrolic ring present in chlorophylls (Huff, 1982). Different reaction mixtures were prepared to verify the activity of POX. Complete mixtures degraded Chl at a constant rate during the first 100 s, but then the reaction rate decreased (Fig. 2a). This is probably due to the diminution of H2O2 concentration, since the replenishment of H2O2
Fig. 1. Changes in the absortion spectra of the complete reaction mixture (enzymatic extract+chlorophyll a+H2O2+p-coumaric acid) after incubation for different times.
restored the initial bleaching rate. No chlorophyll degradation was observed in the absence of H2O2 or the inclusion of boiled extracts, while a low Chl bleaching activity was found in the absence of phenolic cofactors. The effect of H2O2 concentration on chlorophyll-bleaching activity was assayed (Fig. 2b). The chlorophyll-bleaching rate was extremely low in the absence of H2O2, increased to reach a maximum at 1.0 mM and then decreased slightly at 2.5 mM H2O2. Higher concentrations of H2O2 were inhibitory and the activity decreased to zero at 50 mM (data not shown). The inhibitory effect of high concentrations of H2O2 on peroxidase activity has been attributed to damage caused to the heme group present in the enzyme (Sa´nchez-Romero et al., 1995). 2.1. Enzyme characterization Peroxidase-chlorophyll bleaching activities were measured at different temperatures and pHs (Fig. 3a and b). A steep increase in enzyme activity was found when the temperature rose from 20 to 35 C, whereas higher temperatures caused diminution of the activity. The activity at 35 C was nearly twofold higher than at 25 C, the temperature mostly used to measure POX– Chl-bleaching activity (Kuroda et al., 1990; Adachi and Shimokawa, 1995). The optimum pH was determined to be pH 5.2, being 50% of activity at 3.2 and 6.2. The optimum pH range for POX–Chl bleaching activity is lower than 6.0, the optimum pH for POX–guaiacol activity (Civello et al., 1995), suggesting that the value depended on the hydrogen donor. The highest activity was found at an ionic strength of =0.2 (Fig. 3c), similar to that reported for POX obtained from orange flavedo (Huff, 1982).
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
381
Table 1 Apparent kinetic constants of different substrates of the POX bleaching system Compound
Km (mM)
Vmax (mM/min)
Vmax /Km
Chlorophyll a Chlorophyll b Pheophytin a Chlorophyllide a Chlorophyllide b
16.12.0 66.62.1 59.92.4 10.32.0 36.93.8
2.60.2 1.00.1 4.6 0.3 6.10.6 1.30.2
0.161 0.015 0.077 0.588 0.035
into Chl a. The absence of Mg2+ ion in Phe a diminished the affinity for the substrate (higher Km), although the Vmax increased. The removal of the phytol group from chlorophylls a and b increased both the affinity and the Vmax. When catalytic power (Vmax/Km) was calculated, the highest value corresponded to Chld a, which was 5-fold higher than that of Chl a. These results suggest that strawberry fruit POX can preferentially degrade chlorophyll derivatives with the Mg2+ ion present and the phytol group removed. It is worth pointing out that chlorophyllase, the enzyme necessary to remove phytol group from chlorophylls, has been found in strawberry fruit and that the enzyme shows highest affinity for Chl a (Martı´nez et al., 1995). Together, the data suggest that the catabolism of both Chls could start from Chl a in strawberry fruit. 2.3. Effect of inhibitory compounds
Fig. 2. (a) Time courses of chlorophyll a degradation. (~) Reaction mixtures without H2O2. (&) Reaction mixtures with boiled enzymatic extract. (!) Reaction mixtures without p-coumaric acid. (*) Complete reaction mixtures (enzymatic extract+chlorophyll a+H2O2+ p-coumaric acid). (*) Complete reaction mixtures after replenishing H2O2. (b) Effect of (H2O2) on POX-Chl bleaching activity. Vertical bars denotestandard deviation when it exceeds the symbol size.
2.2. Substrate specificity The determination of apparent kinetic parameters (Km and Vmax) was performed by using Chls and their derivatives as substrates (Table 1). The Km value corresponding to Chl a was lower than that of Chl b, while the Vmax was higher, indicating that POX from strawberry fruit has higher affinity for Chl a. Peroxidase extracted from orange flavedo has been assayed with both chlorophylls, and a preferential degradation of Chl a was also reported (Huff, 1982). The fact that Chl b can be transformed in Chl a (Ohtosuka et al., 1997) suggests that Chl b could be degraded after its transformation
Several compounds were analyzed as possible inhibitors of chlorophyll bleaching (Table 2). Although Mn2+ ion has been reported to strongly inhibit POX–Chl bleaching activity in other systems (Adachi and Shimokawa, 1995), the ion at 1 mM concentration caused only a slight inhibition in this study. Compounds with high affinity for the heme group (N3 and CN ) induced a high inhibition, confirming the involvement of peroxidase in Chl bleaching. Free radical scavengers such as hydroquinone, n-propyl gallate, tiron and a-tocopherol inhibited Chl bleaching, suggesting the participation of free radicals as intermediate products. In addition, strong inhibition of Chl bleaching activity was found when reducing agents such as cysteine, dithiothreitol (DTT), glutathione and ascorbic acid were present at 1 mM concentration. In the cases of cysteine, DTT and ascorbic acid, a lag period ranging from 30 to 240 s was observed, after which the Chl bleaching started (data not shown). Higher concentrations of reducing agents induced longer lag periods and a lower POX activity once the reaction started. This low POX activity could be due to the diminution of H2O2 concentration, caused by its consumption during the oxidation of cysteine or DTT. Considering that some plant peroxidases also possess a thiol oxidase activity (Pichorner et al., 1992), another possibility is that the enzyme preferably oxidizes
382
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
Fig. 3. (a) Effect of temperature on POX–Chl bleaching activity. (b) Effect of pH on POX–Chl bleaching activity. (c) Effect of ionic strength of reaction mixture on POX–Chl bleaching activity. (d) Effect of concentration of phenolic compounds on POX–Chl bleaching activity. Vertical bars denote standard deviation when it exceeds the symbol size.
cysteine or DTT instead of chlorophyll. Among the reducing agents tested, the highest inhibition of Chl bleaching was caused by ascorbic acid. It has been reported that ascorbic acid delays the senescence of oat leaves and decreases the chlorophyll loss (Borracino et al., 1994). The content of several antioxidant compounds, including ascorbic acid, decrease during fruit ripening (Baldwin, 1993; Markus et al., 1999). Probably, this compound could inhibit POX ‘in vivo’, therefore delaying the chlorophyll degradation process. During the course of leaf senescence, the antioxidant defense system, including catalase, is impaired while the activity of H2O2-producer enzymes is enhanced, which generates an appropriate environment to increase the chlorophyll bleaching (Hideg, 1997). b-Carotene, which has been
mentioned previously as an inhibitor of Chl bleaching (Kato and Shimizu, 1985), did not show any effect in our system. 2.4. Effect of phenolic compounds As shown in Fig. 2a, POX–Chl bleaching activity is enhanced by the inclusion of p-coumaric acid in the mixture reaction. This compound along with resorcinol has been reported among the most effective phenolic cofactors of peroxidase in the Chl bleaching reaction (Kato and Shimizu, 1985). Therefore, we assayed their effect on POX–Chl bleaching activity present in strawberry fruit (Fig. 3d). Either p-coumaric acid or resorcinol was more effective at a concentration of 3 mM, while
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387 Table 2 Effect of inhibitors on POX–Chl bleaching activity. Different compounds were added to a control mixture reaction (POX extracted from SG fruits+chlorophyll a+H2O2+p-coumaric acid) and the remaining enzyme activity was measured Compound Control reaction (no addition) Mn2+ NaN3 NaCN l-Cysteine DTT Glutathione Ascorbic Acid Hydroquinone n-Propyl gallate Tiron Triethylenediamine a-Tocopherol ß-Carotene a
Concentration (mM)
Activity (pkat)
Inhibition (%)
1 1 0.1 1 1 1 1 1 1 1 5 0.1 0.1
39.03 35.45 11.46 0.36 3.94 8.23 17.19 2.15 0.72 1.79 NDa 39.03 16.47 40.10
0 9.1 70.6 99.0 89.9 78.9 55.9 94.5 98.2 95.4 100 0 57.8 0
ND: not detected.
higher concentrations slightly decreased the activity. In addition, the effect of several phenolic compounds on POX–Chl bleaching activity was analyzed (Table 3). The highest enzyme activities were found with phenol or p-substituted monophenol (p-coumaric acid, phydroxybenzoic acid and p-cresol), in agreement with reports from other systems (Kato and Shimizu, 1985). Low activities were observed with o- or m-substituted monophenol (m-hydroxibenzoic acid and o-cresol), while compounds with o- and p- substitutions in the same compound (e.g. 2,4-dichlorophenol) induced a POX bleaching activity similar to that found with p-monophenol. Among diphenols, only resorcinol (a m-diphenol) was effective to induce Chl bleaching. When o- or p-diphenols or their derivatives were used (catechol, guaiacol, caffeic acid, ellagic acid, hydroquinone and chlorogenic acid), no activity was detected. In the case of catechol, guaiacol, caffeic acid and chlorogenic acid, an enhanced absorption in the blue region of the spectrum was observed, probably due to the phenolic oxidation. Regarding triphenols and derivatives, a mild oxidation of pyrogallol and gallic acid and a short lag initial phase were observed, after which the Chl bleaching reaction started. Among natural coumarines, flavonoids and their derivatives, only umbelliferone and naringenin were effective as cofactors, coincidently with reports in other plant systems (Kato and Shimizu, 1985; Yamauchi and Watada, 1994). Finally, catechin and epicatechin showed oxidation as occurred with other diphenols. When mixtures containing some of the non-effective phenolic compounds (caffeic acid, catechin, ellagic acid, esculin, and quercetin) were added with p-coumaric acid, a measurably enzyme activity was recovered, indicating that their inhibitory effect is reversible (data not shown). In summary, phenolic com-
383
Table 3 Effect of phenolic compounds on POX–Chl bleaching activity. Different phenolic compounds were added to a mixture reaction (POX extracted from SG fruits+chlorophyll a+H2O2+phenolic) and the enzyme activity measured Activity (pkat) Monophenols and derivatives
Phenol 2 mM 2-4 Dichlorophenol 2 mM p-Coumaric acid 2 mM p-Hydroxibenzoic acid 2 mM m-Hydroxibenzoic acid 2 mM o-Cresol 2 mM p-Cresol 2 mM
17.54 12.17 26.14 9.31 0.36 0.36 3.58
Diphenols and derivatives
Catechol 2 mMa Guaiacol 2 mMa Resorcinol 2 mM Caffeic acid 2 mMa Ellagic acid 2 mM Hydroquinone 2 mM Chlorogenic acid 2 mM
NDb ND 13.60 ND ND ND ND
Triphenols and derivatives
Pyrogallol 2 mMa Gallic acid 2 mMa
0.36 7.52
Coumarins and derivatives
Umbelliferone 1.2 mM Eesculin 1.2 mM
3.94 ND
Flavonoids and derivatives
Rutin 0.1 mM Quercetin 0.1 mM Catechin 0.5 mM Epicatechin 0.5 mM Naringenin 1 mM
0.36 ND ND ND 1.07
a b
Phenolic compounds that showed oxidation. ND, not detected.
pounds could act differently probably depending on their chemical structures. Some of the phenolics can activate POX–Chl bleaching while others inhibit the reaction through competition with Chl as substrate or by scavenging free radicals. Strawberry fruit have high concentration of phenolics, which could regulate the activity of POX–Chl bleaching. It has been shown that phenolic compounds inhibit the POX (Zieslin and Ben-Zaken, 1993) and lipoxygenase activity (Richard-Forget et al., 1990), and that they can act as radical scavengers (Rice-Evans et al., 1997). In order to analyze the effect of strawberry endogenous phenolic compounds, we prepared crude extracts with and without PVPP, a phenolic adsorbent, and measured guaiacol–POX and POX–Chl bleaching activities (Table 4). The removal of phenolics by PVPP increased markedly the POX activity with both substrates, suggesting that endogenous phenolic compounds could act as inhibitors. Partial recovery of activity was achieved when extracts were prepared without PVPP and then dialyzed, probably due to the removal of some of the inhibitory compounds. The fact that the enzyme activity was only partially recovered could be due to oxidized phenolicmediated protein modification or to the presence of high
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
384
Table 4 Effect of addition of PVPP in the buffer extraction on guaiacol–POX activity and POX–Chl bleaching activity
With PVPP Without PVPP Without PVPP, dialyzed
Guaiacol (pkat)
Chlorophyll (pkat)
98.4 14.4 16.9 3.1 61.4 9.4
39.033.94 1.07 0.36 22.563.22
molecular mass inhibitors in the extract. According to Huff (1982), these non-dialyzable inhibitory compounds could be polyphenols of high molecular mass. As the amount and composition of phenolic compounds changes during strawberry fruit ripening, we extracted phenolic compounds at several ripening stages and then assayed their effect on POX–Chl bleaching activity (Tables 5 and 6). The concentration of phenolic compounds decreased six-fold during the course of ripening, from 11.4 mg phenol per gram of fruit in ‘small green’ fruit to 1.8 mg phenol per gram of fruit in the ripe stage. Phenolic compounds extracted from fruit at several ripening stages were used to prepare reaction mixtures at the same total phenolics concentration (19 mg/ml). These mixtures were used to test the effect of the endogenous phenolic compounds on POX–Chl bleaching activity. A lag phase was found in all cases, after which the Chl bleaching started and a residual activity could be measured. A shortening of lag phase and an increase of residual activity were found as phenolic extracts from more advanced ripening stages were used.
Therefore, the inhibitory effect of endogenous phenolic compounds on POX–Chl bleaching activity decreases during strawberry fruit ripening, probably due to changes in both the amount and the type of phenolic compounds. These results suggest a role for phenolics as regulators of the POX–Chl bleaching activity during strawberry fruit ripening. Schuster and Herrmann (1985) detected esters of p-coumaric acid and p-hydroxybenzoic acid in strawberry fruit, two of the phenolic compounds that induced POX activity on chlorophyll in this study. As well, ellagic acid, an inhibitor of POX-Chl bleaching, was found in strawberries and its concentration decreased during fruit ripening (Maas et al., 1991). 2.5. Change of chlorophyll content and POX–Chl bleaching activity during ripening Strawberry fruit changes dramatically over a short period during ripening and, once the SG stage is reached, a rapid loss of chlorophylls is observed. The total chlorophyll content per gram of fruit decreased gradually during ripening, with the level of Chl a being higher than that of Chl b at all the stages analyzed (Fig. 4a). Peroxidase chlorophyll bleaching activity was
Table 5 Change in phenolic compound content during strawberry fruit ripening Ripening stage
Concentration of phenolics (mg phenol/g fresh weight)
Small green Large green White Turning Ripe
11.40.5 6.10.4 2.30.1 2.10.1 1.80.1
Table 6 Effect of phenolic extracts on POX–Chl bleaching activity. Phenolic compounds extracted from strawberry fruit at different ripening stages were added to a control mixture reaction (POX extracted from SG fruits+chlorophyll a+H2O2+p-coumaric acid) and the effect on lag phase length and residual activity was measured. The concentration of phenolics added was 19 mg per ml of mixture reaction in all cases Ripening stage
Lag phase (s)
Residual activity (pkat)
Control reaction (no addition) Small green Large green White Turning Ripe
0 150 10 140 10 100 10 75 8 65 7
39.033.94 18.971.07 21.120.72 22.201.07 23.271.07 24.341.07
Fig. 4. (a) Changes in chlorophyll content during ripening of strawberry fruit. Chlorophyl a+b, , Chlorophyll a Chlorophyll b . (b) Changes in POX–Chl bleaching activity during ripening of strawberry fruit. Activity per gram of fruit ; activity per mg of protein . SG: small green; LG: large green; W: white; T: turning; R: red.
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
385
detected in all ripening stages analyzed (Fig. 4b). It was present in SG strawberries before the onset of ripening, increased in LG fruits and then decreased until the end of fruit ripening. ‘In vitro’ POX–Chl bleaching activity is high from SG to W stages, coincidently with the period when most of the fruit chlorophyll is degraded. In addition, we have found a diminution along ripening of inhibitory phenolic compounds on POX–Chl bleaching activity, in agreement with findings reported in citrus (Baldwin, 1993). Together, the results encourage further analysis of POX participation in Chl bleaching during strawberry fruit ripening, although more research is needed to prove the ‘in vivo’ role of peroxidase in chlorophyll degradation.
buffer (pH 5.2), 0.3% (v/v) Triton X-100, 1 mM H2O2, 3 mM p-coumaric acid, 20 mM chlorophyll a (dissolved in acetone) and enzymatic extract, containing 30 mg of total protein, in a final volume of 1 ml. The mixture was incubated at 35 C and the reaction was started by adding H2O2. The degradation of chlorophylls and their derivatives was evaluated by measuring the decrease of OD at: 669 nm for chlorophyll a and chlorophyllide a, 649 nm for chlorophyll b and chlorophyllide b and 672 nm for pheophytin a. Assays at pH different from 6.0 were carried out by adjusting the pH of the mixture with suitable amounts of 1.0 M NaOH, 1.0 M HCl and 0.5 M NaCl to keep the ionic strength constant. Assays at different ionic strengths were carried out by adding suitable amounts of 4 M NaCl to the reaction mixture.
3. Experimental
3.4. Determination of peroxidase–guaiacol activity
3.1. Plant material
The activity of peroxidase using guaiacol as substrate was done according to Civello et al. (1995).
Strawberry fruits (Fragariaananassa, Duch., cv. Selva), grown in a greenhouse, were obtained from local producers. Fruits were harvested and classified according to the external coloration degree and size into different ripening stages: small green (SG), large green (LG), white (W), turning (T) and red (R). Fruits were washed and drained and, after removing calyxes and peduncles, were used immediately or frozen at 60 C.
3.5. Protein dosage Protein concentrations of the extracts were measured by the modified Lowry method described by Potty (1969), measuring OD at 750 nm and using bovine albumin as standard. 3.6. Extraction and measurement of phenolic compounds
3.2. Enzyme extraction Extractions were performed according to Civello et al. (1995) with slight modifications. Fresh or frozen green strawberries (25 g) were homogenized in an Omnimixer homogenizer with 100 ml of the following extraction buffer: 0.02 M Na2HPO4, 0.08 M NaH2PO4, 0.2% (v/v) Triton X-100, 1 M NaCl, 30 g/l polyvinylpolypyrrolidone (PVPP), pH 6.0. The mixture was stirred for 1 h at 4 C and then centrifuged at 9000 g for 20 min at the same temperature. Solid (NH4)2SO4 was added up to 45% saturation to the extract. The mixture was stirred for 5 h at 4 C and then centrifuged at 9000 g for 1 h at the same temperature. The supernatant was treated with (NH4)2SO4 to reach 90% saturation and the resulting mixture was stirred at 4 C overnight. The suspension was centrifuged at 9200 g at 4 C during 1 h. The retained pellet was dissolved in 20 ml of 0.02 M Na2HPO4–0.08 M NaH2PO4 buffer (pH 6.0) and dialyzed against the same buffer at 4 C overnight. 3.3. Determination of peroxidase chlorophyll-bleaching activity Determination of activities was done according to Huff (1982) with slight modifications. The following reaction mixture was used: 0.2 M sodium phosphate
Extractions were performed according to Gil et al. (1997) with slight modifications. Samples of 10 g of frozen strawberries were mixed with 30 ml of acetone and homogenized in an Omnimixer. The mixture was centrifuged at 9000 g for 10 min at 4 C. The pellet was reextracted with 30 ml of acetone and supernatants were pooled (crude extract). This crude extract was extracted three times with 20 ml of hexane to eliminate chlorophylls. The aqueous-acetone phase was evaporated under nitrogen stream at 40 C until 1 ml. Four milliliters of 3% (v/v) formic acid were added to the concentrate and then passed through a C18 Sep-Pak cartridge (Waters), previously activated with 5 ml methanol followed by 5 ml water and then 5 ml of 3% (v/v) formic acid. Phenolics were adsorbed on the column while sugars and other watersoluble compounds were eluted with 10 ml of 3% (v/v) aqueous formic acid. Then, phenolics were recovered with 1.8 ml of 3% (v/v) formic acid in methanol (purified extract). Total phenolic compounds of crude and purified extracts were determined with Folin–Ciocalteau reagent according to Zieslin and Ben-Zaken (1993). Ten microliters of crude extract or a dilution (1:6) of purified extract were mixed with 1.11 ml of water and 200 ml of 1 N Folin–Ciocalteau reagent. After 3 min at 25 C, 1.5 ml of saturated solution of Na2CO3 was added and the reaction mixture was incubated for 1 h at the same
386
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
temperature. The content of phenolic compounds was expressed as milligrams of phenol per g of fresh weight, using phenol as standard. 3.7. Extraction and measurement of chlorophylls Frozen strawberries were homogenized with four volumes of acetone in an Omnimixer. The suspensions were centrifuged at 5000 g for 5 min at 4 C and the chlorophylls (Chls) in the supernatant determined according to Lichtenthaler (1987). 3.8. Preparation of chlorophylls a and b and derivatives Three spinach leaves were homogenized in an Omnimixer with 60 ml of acetone and the resulting suspension was vacuum-filtered through filter paper. The acetone solution obtained was mixed with 50 ml of petroleum ether and 50 ml of saturated NaCl solution to extract the chlorophylls. The ether phase was separated, washed twice with 50 ml of saturated NaCl solution and then evaporated under nitrogen stream. Chlorophylls were redissolved in a solution of 5% (v/v) acetone in hexane and purified by passage through a powdered sucrose column using 5% (v/v) acetone in hexane as mobile phase. Solvents were evaporated under a nitrogen stream and chlorophylls were stored at 20 C until used. Purified chlorophylls were redissolved in acetone for their use. Chl a and Chl b obtained by this procedure were about 98% and 93% pure, respectively. Chlorophyllides (Chld) a and b were prepared from purified Chl a and Chl b by using chlorophyllase obtained from orange flavedo according to Huff (1982). Pheophytin (Phe) a was prepared by treating Chl a with HCl solution, according to Lichtenthaler (1987). References Abeles, F., Takeda, F., 1990. Cellulase activity and ethylene in ripening strawberry and apple fruit. Scientia Horticulturae 42, 269–275. Adachi, M., Shimokawa, K., 1995. Evidence for the involvement of superoxide anion in the ethylene-induced chlorophyll a catabolism of Raphanus sativus cotyledons. Phytochemistry 39, 527–530. Adachi, M., Tsuzuki, E., Shimokawa, K., 1996. Effect of ethylene on degreening of intact radish (Raphanus sativus L) cotyledons. Scientia Horticulturae 65, 1–9. Amir-Shapira, D., Goldschmidt, E., Altman, A., 1987. Chlorophyll catabolism in senescing plant tissues: in vivo breakdown intermediates suggest different degradative pathways for Citrus fruits and parsley leaves. Proceedings of the National Academy of Sciences USA 84, 1901–1905. Baldwin, E., 1993. Citrus fruit. In: Seymour, G., Taylor, J., Tucker, G. (Eds.), Biochemistry of Fruit Ripening. Chapman & Hall, London, pp. 107–149. Borracino, G., Mastropasqua, L., De Leonardis, S., Dipierro, S., 1994. The role of the ascorbic acid system in delaying the senescence of oat (Avena sativa L.) leaf segments. Journal of Plant Physiology 144, 161–166.
Civello, P., Martı´nez, G., Chaves, A., An˜o´n, M., 1995. Peroxidase from strawberry fruit (Fragaria ananassa, Duch.): partial purification and determination of some properties. Journal of Agricultural and Food Chemistry 43, 2596–2601. Gil, M., Holcroft, D., Kader, A., 1997. Changes in strawberry anthocyanins and other polyphenols in response to carbon dioxide treatments. Journal of Agricultural and Food Chemistry 45, 1662–1667. Given, N., Venis, M., Grierson, D., 1988. Hormonal regulation of ripening in the strawberry, a non-climateric fruit. Planta 174, 402– 406. Gross, J., 1982. Changes of chlorophyll and carotenoids in developing strawberry fruits (Fragaria ananassa) cv. Tenira. Gartenbauwissenschaft 47, 142–144. Hideg, E., 1997. Free radical production in photosynthesis under stress conditions. In: Pessarakli, M. (Ed.), Handbook of Photosynthesis. Marcel Dekker, New York, pp. 897–910. Ho¨rtensteiner, S., Wu¨trich, K., Matile, P., Ongania, K., Kra¨utler, B., 1998. The key step in chlorophyll breakdown in higher plants. Journal of Biological Chemistry 273, 15335–15339. Huff, A., 182. Peroxidase-catalysed oxidation of chlorophyll by hydrogen peroxide. Phytochemistry 21, 261–265. Johnson-Flanagan, A., McLachlan, G., 1990. Peroxidase-mediated chlorophyll bleaching in degreening canola (Brassica napus) seeds and its inhibition by sublethal freezing. Physiologia Plantarum 80, 453–459. Johnson-Flanagan, A., Spencer, N., 1996. Chlorophyllase and peroxidase activity during degreening of maturing canola (Brassica napus) and mustard (Brassica juncea) seed. Physiologia Plantarum 97, 353– 359. Kato, M., Shimizu, S., 1985. Chlorophyll metabolism in higher plants VI. Involvement of peroxidase in chlorophyll degradation. Plant Cell Physiology 26, 1291–1301. Kuroda, M., Ozawa, T., Imagawa, H., 1990. Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments. Physiologia Plantarum 80, 555–560. Langmeier, M., Ginsburg, S., Matile, P., 1993. Chlorophyll breakdown in senescent leaves: demonstration of Mg-dechelatase activity. Physiologia Plantarum 89, 347–353. Lichtenthaler, H., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology 148, 350–382. Maas, J., Wang, S., Galletta, G., 1991. Evaluation of strawberry cultivars for ellagic acid content. HortScience 26, 66–68. Maeda, Y., Kurata, H., Adachi, M., Shimokawa, K., 1998. Chlorophyll catabolism in ethylene-treated Citrus unshiu fruits. Journal of the Japanese Society for Horticulturae Science 67, 497–502. Markus, F., Daood, H., Kapitany, J., Biacs, P., 1999. Change in the carotenoid and antioxidant content of spice red pepper (Paprika) as a function of ripening and some technological factors. Journal of Agricultural and Food Chemistry 47, 100–107. Martı´nez, G., Chaves, A., An˜o´n, M., 1994. Effect of gibberellic acid on ripening of strawberry fruits (Fragaria ananassa Duch.). Journal of Plant Growth Regulation 13, 87–91. Martı´nez, G., Civello, P., Chaves, A., An˜o´n, M., 1995. Partial characterization of chlorophyllase from strawberry fruit (Fragaria ananassa, Duch.). Journal of Food Biochemistry 18, 213–226. Matile, P., 1980. Catabolism of chlorophyll. Involvement of peroxidase. Zeitschrift fu¨r Pflanzenphysiologie 99, 475–478. Matile, P., Ho¨rtensteiner, S., Thomas, H., Kra¨utler, B., 1996. Chlorophyll breakdown in senescent leaves. Plant Physiology 112, 1403– 1409. Matile, P., Schellemberg, M., 1996. The cleavage of phaeophorbide a is located in the envelope of barley gerontoplasts. Plant Physiology and Biochemistry 34 (1), 55–59. Matile, P., Ho¨rtensteiner, S., Thomas, H., 1999. Chlorophyll degradation. Annual Review of Plant Physiology and Plant Molecular Biology 50, 67–95.
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387 Ohtosuka, T., Ito, H., Tanaka, A., 1997. Conversion of chlorophyll b to chlorophyll a and the assembly of chlorophyll with apoproteins by isolated chloroplasts. Plant Physiology 113, 137–147. Orthoefer, F., Dugan, L., 1973. The coupled oxidation of chlorophyll a with linoleic acid catalysed by lipoxidase. Journal of the Science of Food and Agriculture 24, 357–365. Perkins-Veazie, P., 1995. Growth and ripening of strawberry fruit. Horticultural Reviews 17, 267–297. Pichorner, H., Couperus, A., Korori, S., Eberman, R., 1992. Plant peroxidase has a thiol oxidase function. Phytochemistry 31, 3371–3376. Potty, V., 1969. Determination of protein in the presence of phenols and pectins. Analytical Biochemistry 29, 535–539. Rice-Evans, C., Miller, N., Paganga, G., 1997. Antioxidant properties of phenolic compounds. Trends in Plant Science 2, 152–159. Richard-Forget, F., Gauillard, F., Hughes, M., Jean-Marc, T., Boivin, P., Nicolas, J., 1990. Inhibition of horse bean and germinated barley lipoxigenases by some phenolic compounds. Journal of Food Science 60, 1325–1329. Sa´nchez-Romero, C., Garcı´a-Go´mez, L., Pliego-Alfaro, F., Heredia, A., 1995. One-step purification of an avocado peroxidase. Plant Physiology and Biochemistry 33, 531–537. Schuster, B., Herrmann, K., 1985. Hydroxybenzoic and hydroxycinnamic acid derivatives in soft fruits. Phytochemistry 24, 2761– 2764.
387
Shioi, Y., Masuda, T., Takamiya, K., Shimokawa, K., 1995. Breakdown of chlorophylls by soluble proteins extracted from leaves of Chenopodium album. Journal of Plant Physiology 145, 416–421. Shioi, Y., Tatsumi, Y., Shimokawa, K., 1991. Enzymatic degradation of chlorophyll in Chenopodium album. Plant Cell Physiology 32, 87– 93. Trebitsh, T., Goldschmidt, E., Riov, J., 1993. Ethylene induces de novo synthesis of chlorophyllase, a chlorophyll degrading enzyme in citrus fruit peel. Proceedings of the National Acadeny of Science USA 90, 9441–9445. Vicentini, F., Iten, F., Matile, P., 1995. Development of an assay for Mg-dechelatase of oilseed rape cotyledons, using chlorophyllin as the substrate. Physiologia Plantarum 94, 57–63. Watanabe, K., Ohta, H., Tsuchiya, T., Mikami, B., Masuda, T., Shioi, Y., Takamiya, K., 1999. Purification and some properties of pheophorbidase in Chenopodium album. Plant Cell Physiology 40, 104– 108. Yamauchi, N., Watada, A., 1994. Effectiveness of various phenolic compounds in degradation of chlorophyll by in vitro peroxidasehyrogen peroxide system. Journal of the Japanese Society for Horticultural Science 63, 439–444. Zieslin, N., Ben-Zaken, R., 1993. Peroxidase activity and presence of phenolic substances in peduncles of rose flowers. Plant Physiology and Biochemistry 31, 333–339.
Characterization of peroxidase-mediated chlorophyll bleaching in strawberry fruit Gustavo A. Martı´neza,*, Pedro M. Civelloa, Alicia R. Chavesb, Marı´a C. An˜o´nb a
Instituto de Investigaciones Biotecnolo´gicas-Instituto Tecnolo´gico de Chascomu´s (IIB-INTECH), UNSAM-CONICET, Camino de Circunvalacio´n Laguna Km 6, 7130 Chascomu´s, Argentina b Centro de Investigacio´n y Desarrollo en Criotecnologı´a de Alimentos (CIDCA), Facultad de Ciencias Exactas, UNLP, CONICET, 47 y 116, 1900 La Plata, Argentina Received 2 January 2001; received in revised form 9 May 2001
Abstract Peroxidase (POX) from strawberry fruits was analyzed for its capacity to bleach chlorophyll. The partially purified enzyme preperation catalyzed the bleaching of chlorophylls and their derivatives in the presence of H2O2 and phenolic compounds. The optimal reaction conditions were 35 C, pH 5.2 and ionic strength equal to 0.2. The maximum activity was observed at 1 mM of H2O2, while higher concentrations inhibited enzyme activity. Compounds with a high affinity to the heme group, radical scavengers and reducing agents, showed an inhibitory effect. Phenolic compounds such as umbelliferone, naringenin and p-substituted monophenols acted as cofactors. Instead, other phenolic compounds tested such as caffeic acid, catechin, ellagic acid, esculin and quercetin inhibited the activity of POX on chlorophylls. Phenolic compounds extracted from strawberry fruits showed an inhibitory effect on POX-chlorophyll bleaching activity, although this effect decreased markedly during ripening. POX showed higher affinity for compounds derived from chlorophyll a than from chlorophyll b, and the enzyme preferentially degraded chlorophyll derivatives with the Mg2+ ion present and the phytol group removed. The POX-chlorophyll bleaching activity was found in all ripening stages from small green to ripe, the highest activity corresponding to large green fruits. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fragaria x ananassa; Rosaceae; Strawberry; Chlorophyll; Peroxidase; Phenolic compounds
1. Introduction Chlorophyll (Chl) degradation is a characteristic symptom of leaf senescence and fruit ripening. During senescence, chlorophyll-protein complexes are disassembled and the pigments liberated. Free chlorophylls (Chls) are highly photoactive compounds that can generate free radicals, causing damage to cell membranes (Matile et al., 1996). It has been suggested that two types of reactions are involved in Chl catabolism (Shioi et al., 1995). Type I reactions include the loss of phytol, Mg2+, and other modifications of the side chains that do not involve the breaking of the tetrapyrrolic ring present in chlorophylls. Chlorophyllase, which removes the phytol and produces chlorophyllides (Chlds), has been involved in * Corresponding author. Postal address: Camino de Circunvalacion Laguna Km 6, (7130) Chascomus, Argentina. Tel.: +54-2241424049; fax: +54-2241-424048. E-mail address: [email protected] (G.A. Martı´nez).
the first step of Chl breakdown (Trebitsh et al., 1993; Matile et al., 1996). Another enzyme involved in chlorophyll catabolism is Mg-dechelatase, which eliminates Mg2+ from Chl or Chld to produce pheophytin (Phe) or pheophorbide (Pheo), respectively (Langmeier et al., 1993; Vicentini et al., 1995). Further catabolism of Pheo involves the elimination of –COOCH3 to form pyropheophorbide, and oxidation to form hydroxypheophorbide (Shioi et al., 1991) or the formation of C132-carboxylpyropheophorbide by the action of pheophorbidase (Watanabe et al., 1999). Type II reactions involve the oxidative cleavage (bleaching) of the tetrapyrrole ring. It has been reported that the cleavage of Phe a by a pheophorbide a oxygenase is the key regulatory step in the Chl catabolism pathway (Matile and Schellemberg, 1996; Ho¨rtensteiner et al., 1998; Matile et al., 1999). However, other oxidative enzymes such as lipoxygenase (Orthoefer and Dugan, 1973), Chl oxidase, and peroxidase also appear to be involved in Chl bleaching.
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00266-7
380
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
Peroxidase (POX) is a ubiquitous enzyme located in several subcellular compartments, including chloroplasts (Johnson-Flanagan and McLachlan, 1990; Kuroda et al., 1990). Although the role of POX in Chl degradation ‘in vivo’ is controversial (Matile et al., 1999), many studies have supported its possible participation in Chl catabolism (Matile, 1980; Huff, 1982; Johnson-Flanagan and McLachlan, 1990; Johnson-Flanagan and Spencer; 1996; Maeda et al., 1998). Probably, the controversy has been caused by the existence of different chlorophyll degradation pathways (Amir-Shapira et al., 1987; Trebitsh et al., 1993) and the presence of multiple POX isoenzymes with uneven capacities to degrade chlorophylls (Kuroda et al., 1990; Adachi et al., 1996). Strawberry is classified as a non-climacteric fruit, and neither the respiration rate nor ethylene production increase as the fruit changes its color, texture and flavor (Abeles and Takeda, 1990). The chlorophyll content diminishes very quickly as the fruit ripens (Given et al., 1988; Martı´nez et al., 1994; Perkins-Veazie, 1995), and the chloroplasts disintegrate during the process (Gross, 1982; Perkins-Veazie, 1995) without conversion into chromoplasts or gerontoplasts, as occurs in tomato fruits and leaves respectively (Matile et al., 1996). These characteristics make strawberry fruit an interesting model system in which to study chlorophyll degradation during fruit ripening. In the present work, we have partially characterized the activity of strawberry peroxidase for bleaching chlorophyll, determined the influence of phenolic compounds on this activity and analyzed the change of POX bleaching activity during ripening.
2. Results and discussion We have previously reported the presence of two cationic peroxidase isoenzymes in strawberry fruit, both of which are associated with membranes and show maximum activity at the green ripening stage (Civello et al., 1995). It has been suggested that cationic peroxidases, located in the chloroplast, are involved in Chl bleaching (Kuroda et al., 1990). In order to analyze strawberry POX activity on chlorophylls, extracts from green fruits were prepared and partially purified by means of (NH4)2SO4 fractionation. The absorption spectrum of complete reaction mixtures (enzyme extract, chlorophyll, H2O2, and p-coumaric acid) showed a progressive reduction of the absorbance at 669 nm (Fig. 1). According to previous reports, this effect is due to the cleavage of the tetrapyrrolic ring present in chlorophylls (Huff, 1982). Different reaction mixtures were prepared to verify the activity of POX. Complete mixtures degraded Chl at a constant rate during the first 100 s, but then the reaction rate decreased (Fig. 2a). This is probably due to the diminution of H2O2 concentration, since the replenishment of H2O2
Fig. 1. Changes in the absortion spectra of the complete reaction mixture (enzymatic extract+chlorophyll a+H2O2+p-coumaric acid) after incubation for different times.
restored the initial bleaching rate. No chlorophyll degradation was observed in the absence of H2O2 or the inclusion of boiled extracts, while a low Chl bleaching activity was found in the absence of phenolic cofactors. The effect of H2O2 concentration on chlorophyll-bleaching activity was assayed (Fig. 2b). The chlorophyll-bleaching rate was extremely low in the absence of H2O2, increased to reach a maximum at 1.0 mM and then decreased slightly at 2.5 mM H2O2. Higher concentrations of H2O2 were inhibitory and the activity decreased to zero at 50 mM (data not shown). The inhibitory effect of high concentrations of H2O2 on peroxidase activity has been attributed to damage caused to the heme group present in the enzyme (Sa´nchez-Romero et al., 1995). 2.1. Enzyme characterization Peroxidase-chlorophyll bleaching activities were measured at different temperatures and pHs (Fig. 3a and b). A steep increase in enzyme activity was found when the temperature rose from 20 to 35 C, whereas higher temperatures caused diminution of the activity. The activity at 35 C was nearly twofold higher than at 25 C, the temperature mostly used to measure POX– Chl-bleaching activity (Kuroda et al., 1990; Adachi and Shimokawa, 1995). The optimum pH was determined to be pH 5.2, being 50% of activity at 3.2 and 6.2. The optimum pH range for POX–Chl bleaching activity is lower than 6.0, the optimum pH for POX–guaiacol activity (Civello et al., 1995), suggesting that the value depended on the hydrogen donor. The highest activity was found at an ionic strength of =0.2 (Fig. 3c), similar to that reported for POX obtained from orange flavedo (Huff, 1982).
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
381
Table 1 Apparent kinetic constants of different substrates of the POX bleaching system Compound
Km (mM)
Vmax (mM/min)
Vmax /Km
Chlorophyll a Chlorophyll b Pheophytin a Chlorophyllide a Chlorophyllide b
16.12.0 66.62.1 59.92.4 10.32.0 36.93.8
2.60.2 1.00.1 4.6 0.3 6.10.6 1.30.2
0.161 0.015 0.077 0.588 0.035
into Chl a. The absence of Mg2+ ion in Phe a diminished the affinity for the substrate (higher Km), although the Vmax increased. The removal of the phytol group from chlorophylls a and b increased both the affinity and the Vmax. When catalytic power (Vmax/Km) was calculated, the highest value corresponded to Chld a, which was 5-fold higher than that of Chl a. These results suggest that strawberry fruit POX can preferentially degrade chlorophyll derivatives with the Mg2+ ion present and the phytol group removed. It is worth pointing out that chlorophyllase, the enzyme necessary to remove phytol group from chlorophylls, has been found in strawberry fruit and that the enzyme shows highest affinity for Chl a (Martı´nez et al., 1995). Together, the data suggest that the catabolism of both Chls could start from Chl a in strawberry fruit. 2.3. Effect of inhibitory compounds
Fig. 2. (a) Time courses of chlorophyll a degradation. (~) Reaction mixtures without H2O2. (&) Reaction mixtures with boiled enzymatic extract. (!) Reaction mixtures without p-coumaric acid. (*) Complete reaction mixtures (enzymatic extract+chlorophyll a+H2O2+ p-coumaric acid). (*) Complete reaction mixtures after replenishing H2O2. (b) Effect of (H2O2) on POX-Chl bleaching activity. Vertical bars denotestandard deviation when it exceeds the symbol size.
2.2. Substrate specificity The determination of apparent kinetic parameters (Km and Vmax) was performed by using Chls and their derivatives as substrates (Table 1). The Km value corresponding to Chl a was lower than that of Chl b, while the Vmax was higher, indicating that POX from strawberry fruit has higher affinity for Chl a. Peroxidase extracted from orange flavedo has been assayed with both chlorophylls, and a preferential degradation of Chl a was also reported (Huff, 1982). The fact that Chl b can be transformed in Chl a (Ohtosuka et al., 1997) suggests that Chl b could be degraded after its transformation
Several compounds were analyzed as possible inhibitors of chlorophyll bleaching (Table 2). Although Mn2+ ion has been reported to strongly inhibit POX–Chl bleaching activity in other systems (Adachi and Shimokawa, 1995), the ion at 1 mM concentration caused only a slight inhibition in this study. Compounds with high affinity for the heme group (N3 and CN ) induced a high inhibition, confirming the involvement of peroxidase in Chl bleaching. Free radical scavengers such as hydroquinone, n-propyl gallate, tiron and a-tocopherol inhibited Chl bleaching, suggesting the participation of free radicals as intermediate products. In addition, strong inhibition of Chl bleaching activity was found when reducing agents such as cysteine, dithiothreitol (DTT), glutathione and ascorbic acid were present at 1 mM concentration. In the cases of cysteine, DTT and ascorbic acid, a lag period ranging from 30 to 240 s was observed, after which the Chl bleaching started (data not shown). Higher concentrations of reducing agents induced longer lag periods and a lower POX activity once the reaction started. This low POX activity could be due to the diminution of H2O2 concentration, caused by its consumption during the oxidation of cysteine or DTT. Considering that some plant peroxidases also possess a thiol oxidase activity (Pichorner et al., 1992), another possibility is that the enzyme preferably oxidizes
382
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
Fig. 3. (a) Effect of temperature on POX–Chl bleaching activity. (b) Effect of pH on POX–Chl bleaching activity. (c) Effect of ionic strength of reaction mixture on POX–Chl bleaching activity. (d) Effect of concentration of phenolic compounds on POX–Chl bleaching activity. Vertical bars denote standard deviation when it exceeds the symbol size.
cysteine or DTT instead of chlorophyll. Among the reducing agents tested, the highest inhibition of Chl bleaching was caused by ascorbic acid. It has been reported that ascorbic acid delays the senescence of oat leaves and decreases the chlorophyll loss (Borracino et al., 1994). The content of several antioxidant compounds, including ascorbic acid, decrease during fruit ripening (Baldwin, 1993; Markus et al., 1999). Probably, this compound could inhibit POX ‘in vivo’, therefore delaying the chlorophyll degradation process. During the course of leaf senescence, the antioxidant defense system, including catalase, is impaired while the activity of H2O2-producer enzymes is enhanced, which generates an appropriate environment to increase the chlorophyll bleaching (Hideg, 1997). b-Carotene, which has been
mentioned previously as an inhibitor of Chl bleaching (Kato and Shimizu, 1985), did not show any effect in our system. 2.4. Effect of phenolic compounds As shown in Fig. 2a, POX–Chl bleaching activity is enhanced by the inclusion of p-coumaric acid in the mixture reaction. This compound along with resorcinol has been reported among the most effective phenolic cofactors of peroxidase in the Chl bleaching reaction (Kato and Shimizu, 1985). Therefore, we assayed their effect on POX–Chl bleaching activity present in strawberry fruit (Fig. 3d). Either p-coumaric acid or resorcinol was more effective at a concentration of 3 mM, while
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387 Table 2 Effect of inhibitors on POX–Chl bleaching activity. Different compounds were added to a control mixture reaction (POX extracted from SG fruits+chlorophyll a+H2O2+p-coumaric acid) and the remaining enzyme activity was measured Compound Control reaction (no addition) Mn2+ NaN3 NaCN l-Cysteine DTT Glutathione Ascorbic Acid Hydroquinone n-Propyl gallate Tiron Triethylenediamine a-Tocopherol ß-Carotene a
Concentration (mM)
Activity (pkat)
Inhibition (%)
1 1 0.1 1 1 1 1 1 1 1 5 0.1 0.1
39.03 35.45 11.46 0.36 3.94 8.23 17.19 2.15 0.72 1.79 NDa 39.03 16.47 40.10
0 9.1 70.6 99.0 89.9 78.9 55.9 94.5 98.2 95.4 100 0 57.8 0
ND: not detected.
higher concentrations slightly decreased the activity. In addition, the effect of several phenolic compounds on POX–Chl bleaching activity was analyzed (Table 3). The highest enzyme activities were found with phenol or p-substituted monophenol (p-coumaric acid, phydroxybenzoic acid and p-cresol), in agreement with reports from other systems (Kato and Shimizu, 1985). Low activities were observed with o- or m-substituted monophenol (m-hydroxibenzoic acid and o-cresol), while compounds with o- and p- substitutions in the same compound (e.g. 2,4-dichlorophenol) induced a POX bleaching activity similar to that found with p-monophenol. Among diphenols, only resorcinol (a m-diphenol) was effective to induce Chl bleaching. When o- or p-diphenols or their derivatives were used (catechol, guaiacol, caffeic acid, ellagic acid, hydroquinone and chlorogenic acid), no activity was detected. In the case of catechol, guaiacol, caffeic acid and chlorogenic acid, an enhanced absorption in the blue region of the spectrum was observed, probably due to the phenolic oxidation. Regarding triphenols and derivatives, a mild oxidation of pyrogallol and gallic acid and a short lag initial phase were observed, after which the Chl bleaching reaction started. Among natural coumarines, flavonoids and their derivatives, only umbelliferone and naringenin were effective as cofactors, coincidently with reports in other plant systems (Kato and Shimizu, 1985; Yamauchi and Watada, 1994). Finally, catechin and epicatechin showed oxidation as occurred with other diphenols. When mixtures containing some of the non-effective phenolic compounds (caffeic acid, catechin, ellagic acid, esculin, and quercetin) were added with p-coumaric acid, a measurably enzyme activity was recovered, indicating that their inhibitory effect is reversible (data not shown). In summary, phenolic com-
383
Table 3 Effect of phenolic compounds on POX–Chl bleaching activity. Different phenolic compounds were added to a mixture reaction (POX extracted from SG fruits+chlorophyll a+H2O2+phenolic) and the enzyme activity measured Activity (pkat) Monophenols and derivatives
Phenol 2 mM 2-4 Dichlorophenol 2 mM p-Coumaric acid 2 mM p-Hydroxibenzoic acid 2 mM m-Hydroxibenzoic acid 2 mM o-Cresol 2 mM p-Cresol 2 mM
17.54 12.17 26.14 9.31 0.36 0.36 3.58
Diphenols and derivatives
Catechol 2 mMa Guaiacol 2 mMa Resorcinol 2 mM Caffeic acid 2 mMa Ellagic acid 2 mM Hydroquinone 2 mM Chlorogenic acid 2 mM
NDb ND 13.60 ND ND ND ND
Triphenols and derivatives
Pyrogallol 2 mMa Gallic acid 2 mMa
0.36 7.52
Coumarins and derivatives
Umbelliferone 1.2 mM Eesculin 1.2 mM
3.94 ND
Flavonoids and derivatives
Rutin 0.1 mM Quercetin 0.1 mM Catechin 0.5 mM Epicatechin 0.5 mM Naringenin 1 mM
0.36 ND ND ND 1.07
a b
Phenolic compounds that showed oxidation. ND, not detected.
pounds could act differently probably depending on their chemical structures. Some of the phenolics can activate POX–Chl bleaching while others inhibit the reaction through competition with Chl as substrate or by scavenging free radicals. Strawberry fruit have high concentration of phenolics, which could regulate the activity of POX–Chl bleaching. It has been shown that phenolic compounds inhibit the POX (Zieslin and Ben-Zaken, 1993) and lipoxygenase activity (Richard-Forget et al., 1990), and that they can act as radical scavengers (Rice-Evans et al., 1997). In order to analyze the effect of strawberry endogenous phenolic compounds, we prepared crude extracts with and without PVPP, a phenolic adsorbent, and measured guaiacol–POX and POX–Chl bleaching activities (Table 4). The removal of phenolics by PVPP increased markedly the POX activity with both substrates, suggesting that endogenous phenolic compounds could act as inhibitors. Partial recovery of activity was achieved when extracts were prepared without PVPP and then dialyzed, probably due to the removal of some of the inhibitory compounds. The fact that the enzyme activity was only partially recovered could be due to oxidized phenolicmediated protein modification or to the presence of high
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
384
Table 4 Effect of addition of PVPP in the buffer extraction on guaiacol–POX activity and POX–Chl bleaching activity
With PVPP Without PVPP Without PVPP, dialyzed
Guaiacol (pkat)
Chlorophyll (pkat)
98.4 14.4 16.9 3.1 61.4 9.4
39.033.94 1.07 0.36 22.563.22
molecular mass inhibitors in the extract. According to Huff (1982), these non-dialyzable inhibitory compounds could be polyphenols of high molecular mass. As the amount and composition of phenolic compounds changes during strawberry fruit ripening, we extracted phenolic compounds at several ripening stages and then assayed their effect on POX–Chl bleaching activity (Tables 5 and 6). The concentration of phenolic compounds decreased six-fold during the course of ripening, from 11.4 mg phenol per gram of fruit in ‘small green’ fruit to 1.8 mg phenol per gram of fruit in the ripe stage. Phenolic compounds extracted from fruit at several ripening stages were used to prepare reaction mixtures at the same total phenolics concentration (19 mg/ml). These mixtures were used to test the effect of the endogenous phenolic compounds on POX–Chl bleaching activity. A lag phase was found in all cases, after which the Chl bleaching started and a residual activity could be measured. A shortening of lag phase and an increase of residual activity were found as phenolic extracts from more advanced ripening stages were used.
Therefore, the inhibitory effect of endogenous phenolic compounds on POX–Chl bleaching activity decreases during strawberry fruit ripening, probably due to changes in both the amount and the type of phenolic compounds. These results suggest a role for phenolics as regulators of the POX–Chl bleaching activity during strawberry fruit ripening. Schuster and Herrmann (1985) detected esters of p-coumaric acid and p-hydroxybenzoic acid in strawberry fruit, two of the phenolic compounds that induced POX activity on chlorophyll in this study. As well, ellagic acid, an inhibitor of POX-Chl bleaching, was found in strawberries and its concentration decreased during fruit ripening (Maas et al., 1991). 2.5. Change of chlorophyll content and POX–Chl bleaching activity during ripening Strawberry fruit changes dramatically over a short period during ripening and, once the SG stage is reached, a rapid loss of chlorophylls is observed. The total chlorophyll content per gram of fruit decreased gradually during ripening, with the level of Chl a being higher than that of Chl b at all the stages analyzed (Fig. 4a). Peroxidase chlorophyll bleaching activity was
Table 5 Change in phenolic compound content during strawberry fruit ripening Ripening stage
Concentration of phenolics (mg phenol/g fresh weight)
Small green Large green White Turning Ripe
11.40.5 6.10.4 2.30.1 2.10.1 1.80.1
Table 6 Effect of phenolic extracts on POX–Chl bleaching activity. Phenolic compounds extracted from strawberry fruit at different ripening stages were added to a control mixture reaction (POX extracted from SG fruits+chlorophyll a+H2O2+p-coumaric acid) and the effect on lag phase length and residual activity was measured. The concentration of phenolics added was 19 mg per ml of mixture reaction in all cases Ripening stage
Lag phase (s)
Residual activity (pkat)
Control reaction (no addition) Small green Large green White Turning Ripe
0 150 10 140 10 100 10 75 8 65 7
39.033.94 18.971.07 21.120.72 22.201.07 23.271.07 24.341.07
Fig. 4. (a) Changes in chlorophyll content during ripening of strawberry fruit. Chlorophyl a+b, , Chlorophyll a Chlorophyll b . (b) Changes in POX–Chl bleaching activity during ripening of strawberry fruit. Activity per gram of fruit ; activity per mg of protein . SG: small green; LG: large green; W: white; T: turning; R: red.
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
385
detected in all ripening stages analyzed (Fig. 4b). It was present in SG strawberries before the onset of ripening, increased in LG fruits and then decreased until the end of fruit ripening. ‘In vitro’ POX–Chl bleaching activity is high from SG to W stages, coincidently with the period when most of the fruit chlorophyll is degraded. In addition, we have found a diminution along ripening of inhibitory phenolic compounds on POX–Chl bleaching activity, in agreement with findings reported in citrus (Baldwin, 1993). Together, the results encourage further analysis of POX participation in Chl bleaching during strawberry fruit ripening, although more research is needed to prove the ‘in vivo’ role of peroxidase in chlorophyll degradation.
buffer (pH 5.2), 0.3% (v/v) Triton X-100, 1 mM H2O2, 3 mM p-coumaric acid, 20 mM chlorophyll a (dissolved in acetone) and enzymatic extract, containing 30 mg of total protein, in a final volume of 1 ml. The mixture was incubated at 35 C and the reaction was started by adding H2O2. The degradation of chlorophylls and their derivatives was evaluated by measuring the decrease of OD at: 669 nm for chlorophyll a and chlorophyllide a, 649 nm for chlorophyll b and chlorophyllide b and 672 nm for pheophytin a. Assays at pH different from 6.0 were carried out by adjusting the pH of the mixture with suitable amounts of 1.0 M NaOH, 1.0 M HCl and 0.5 M NaCl to keep the ionic strength constant. Assays at different ionic strengths were carried out by adding suitable amounts of 4 M NaCl to the reaction mixture.
3. Experimental
3.4. Determination of peroxidase–guaiacol activity
3.1. Plant material
The activity of peroxidase using guaiacol as substrate was done according to Civello et al. (1995).
Strawberry fruits (Fragariaananassa, Duch., cv. Selva), grown in a greenhouse, were obtained from local producers. Fruits were harvested and classified according to the external coloration degree and size into different ripening stages: small green (SG), large green (LG), white (W), turning (T) and red (R). Fruits were washed and drained and, after removing calyxes and peduncles, were used immediately or frozen at 60 C.
3.5. Protein dosage Protein concentrations of the extracts were measured by the modified Lowry method described by Potty (1969), measuring OD at 750 nm and using bovine albumin as standard. 3.6. Extraction and measurement of phenolic compounds
3.2. Enzyme extraction Extractions were performed according to Civello et al. (1995) with slight modifications. Fresh or frozen green strawberries (25 g) were homogenized in an Omnimixer homogenizer with 100 ml of the following extraction buffer: 0.02 M Na2HPO4, 0.08 M NaH2PO4, 0.2% (v/v) Triton X-100, 1 M NaCl, 30 g/l polyvinylpolypyrrolidone (PVPP), pH 6.0. The mixture was stirred for 1 h at 4 C and then centrifuged at 9000 g for 20 min at the same temperature. Solid (NH4)2SO4 was added up to 45% saturation to the extract. The mixture was stirred for 5 h at 4 C and then centrifuged at 9000 g for 1 h at the same temperature. The supernatant was treated with (NH4)2SO4 to reach 90% saturation and the resulting mixture was stirred at 4 C overnight. The suspension was centrifuged at 9200 g at 4 C during 1 h. The retained pellet was dissolved in 20 ml of 0.02 M Na2HPO4–0.08 M NaH2PO4 buffer (pH 6.0) and dialyzed against the same buffer at 4 C overnight. 3.3. Determination of peroxidase chlorophyll-bleaching activity Determination of activities was done according to Huff (1982) with slight modifications. The following reaction mixture was used: 0.2 M sodium phosphate
Extractions were performed according to Gil et al. (1997) with slight modifications. Samples of 10 g of frozen strawberries were mixed with 30 ml of acetone and homogenized in an Omnimixer. The mixture was centrifuged at 9000 g for 10 min at 4 C. The pellet was reextracted with 30 ml of acetone and supernatants were pooled (crude extract). This crude extract was extracted three times with 20 ml of hexane to eliminate chlorophylls. The aqueous-acetone phase was evaporated under nitrogen stream at 40 C until 1 ml. Four milliliters of 3% (v/v) formic acid were added to the concentrate and then passed through a C18 Sep-Pak cartridge (Waters), previously activated with 5 ml methanol followed by 5 ml water and then 5 ml of 3% (v/v) formic acid. Phenolics were adsorbed on the column while sugars and other watersoluble compounds were eluted with 10 ml of 3% (v/v) aqueous formic acid. Then, phenolics were recovered with 1.8 ml of 3% (v/v) formic acid in methanol (purified extract). Total phenolic compounds of crude and purified extracts were determined with Folin–Ciocalteau reagent according to Zieslin and Ben-Zaken (1993). Ten microliters of crude extract or a dilution (1:6) of purified extract were mixed with 1.11 ml of water and 200 ml of 1 N Folin–Ciocalteau reagent. After 3 min at 25 C, 1.5 ml of saturated solution of Na2CO3 was added and the reaction mixture was incubated for 1 h at the same
386
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387
temperature. The content of phenolic compounds was expressed as milligrams of phenol per g of fresh weight, using phenol as standard. 3.7. Extraction and measurement of chlorophylls Frozen strawberries were homogenized with four volumes of acetone in an Omnimixer. The suspensions were centrifuged at 5000 g for 5 min at 4 C and the chlorophylls (Chls) in the supernatant determined according to Lichtenthaler (1987). 3.8. Preparation of chlorophylls a and b and derivatives Three spinach leaves were homogenized in an Omnimixer with 60 ml of acetone and the resulting suspension was vacuum-filtered through filter paper. The acetone solution obtained was mixed with 50 ml of petroleum ether and 50 ml of saturated NaCl solution to extract the chlorophylls. The ether phase was separated, washed twice with 50 ml of saturated NaCl solution and then evaporated under nitrogen stream. Chlorophylls were redissolved in a solution of 5% (v/v) acetone in hexane and purified by passage through a powdered sucrose column using 5% (v/v) acetone in hexane as mobile phase. Solvents were evaporated under a nitrogen stream and chlorophylls were stored at 20 C until used. Purified chlorophylls were redissolved in acetone for their use. Chl a and Chl b obtained by this procedure were about 98% and 93% pure, respectively. Chlorophyllides (Chld) a and b were prepared from purified Chl a and Chl b by using chlorophyllase obtained from orange flavedo according to Huff (1982). Pheophytin (Phe) a was prepared by treating Chl a with HCl solution, according to Lichtenthaler (1987). References Abeles, F., Takeda, F., 1990. Cellulase activity and ethylene in ripening strawberry and apple fruit. Scientia Horticulturae 42, 269–275. Adachi, M., Shimokawa, K., 1995. Evidence for the involvement of superoxide anion in the ethylene-induced chlorophyll a catabolism of Raphanus sativus cotyledons. Phytochemistry 39, 527–530. Adachi, M., Tsuzuki, E., Shimokawa, K., 1996. Effect of ethylene on degreening of intact radish (Raphanus sativus L) cotyledons. Scientia Horticulturae 65, 1–9. Amir-Shapira, D., Goldschmidt, E., Altman, A., 1987. Chlorophyll catabolism in senescing plant tissues: in vivo breakdown intermediates suggest different degradative pathways for Citrus fruits and parsley leaves. Proceedings of the National Academy of Sciences USA 84, 1901–1905. Baldwin, E., 1993. Citrus fruit. In: Seymour, G., Taylor, J., Tucker, G. (Eds.), Biochemistry of Fruit Ripening. Chapman & Hall, London, pp. 107–149. Borracino, G., Mastropasqua, L., De Leonardis, S., Dipierro, S., 1994. The role of the ascorbic acid system in delaying the senescence of oat (Avena sativa L.) leaf segments. Journal of Plant Physiology 144, 161–166.
Civello, P., Martı´nez, G., Chaves, A., An˜o´n, M., 1995. Peroxidase from strawberry fruit (Fragaria ananassa, Duch.): partial purification and determination of some properties. Journal of Agricultural and Food Chemistry 43, 2596–2601. Gil, M., Holcroft, D., Kader, A., 1997. Changes in strawberry anthocyanins and other polyphenols in response to carbon dioxide treatments. Journal of Agricultural and Food Chemistry 45, 1662–1667. Given, N., Venis, M., Grierson, D., 1988. Hormonal regulation of ripening in the strawberry, a non-climateric fruit. Planta 174, 402– 406. Gross, J., 1982. Changes of chlorophyll and carotenoids in developing strawberry fruits (Fragaria ananassa) cv. Tenira. Gartenbauwissenschaft 47, 142–144. Hideg, E., 1997. Free radical production in photosynthesis under stress conditions. In: Pessarakli, M. (Ed.), Handbook of Photosynthesis. Marcel Dekker, New York, pp. 897–910. Ho¨rtensteiner, S., Wu¨trich, K., Matile, P., Ongania, K., Kra¨utler, B., 1998. The key step in chlorophyll breakdown in higher plants. Journal of Biological Chemistry 273, 15335–15339. Huff, A., 182. Peroxidase-catalysed oxidation of chlorophyll by hydrogen peroxide. Phytochemistry 21, 261–265. Johnson-Flanagan, A., McLachlan, G., 1990. Peroxidase-mediated chlorophyll bleaching in degreening canola (Brassica napus) seeds and its inhibition by sublethal freezing. Physiologia Plantarum 80, 453–459. Johnson-Flanagan, A., Spencer, N., 1996. Chlorophyllase and peroxidase activity during degreening of maturing canola (Brassica napus) and mustard (Brassica juncea) seed. Physiologia Plantarum 97, 353– 359. Kato, M., Shimizu, S., 1985. Chlorophyll metabolism in higher plants VI. Involvement of peroxidase in chlorophyll degradation. Plant Cell Physiology 26, 1291–1301. Kuroda, M., Ozawa, T., Imagawa, H., 1990. Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments. Physiologia Plantarum 80, 555–560. Langmeier, M., Ginsburg, S., Matile, P., 1993. Chlorophyll breakdown in senescent leaves: demonstration of Mg-dechelatase activity. Physiologia Plantarum 89, 347–353. Lichtenthaler, H., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology 148, 350–382. Maas, J., Wang, S., Galletta, G., 1991. Evaluation of strawberry cultivars for ellagic acid content. HortScience 26, 66–68. Maeda, Y., Kurata, H., Adachi, M., Shimokawa, K., 1998. Chlorophyll catabolism in ethylene-treated Citrus unshiu fruits. Journal of the Japanese Society for Horticulturae Science 67, 497–502. Markus, F., Daood, H., Kapitany, J., Biacs, P., 1999. Change in the carotenoid and antioxidant content of spice red pepper (Paprika) as a function of ripening and some technological factors. Journal of Agricultural and Food Chemistry 47, 100–107. Martı´nez, G., Chaves, A., An˜o´n, M., 1994. Effect of gibberellic acid on ripening of strawberry fruits (Fragaria ananassa Duch.). Journal of Plant Growth Regulation 13, 87–91. Martı´nez, G., Civello, P., Chaves, A., An˜o´n, M., 1995. Partial characterization of chlorophyllase from strawberry fruit (Fragaria ananassa, Duch.). Journal of Food Biochemistry 18, 213–226. Matile, P., 1980. Catabolism of chlorophyll. Involvement of peroxidase. Zeitschrift fu¨r Pflanzenphysiologie 99, 475–478. Matile, P., Ho¨rtensteiner, S., Thomas, H., Kra¨utler, B., 1996. Chlorophyll breakdown in senescent leaves. Plant Physiology 112, 1403– 1409. Matile, P., Schellemberg, M., 1996. The cleavage of phaeophorbide a is located in the envelope of barley gerontoplasts. Plant Physiology and Biochemistry 34 (1), 55–59. Matile, P., Ho¨rtensteiner, S., Thomas, H., 1999. Chlorophyll degradation. Annual Review of Plant Physiology and Plant Molecular Biology 50, 67–95.
G.A. Martı´nez et al. / Phytochemistry 58 (2001) 379–387 Ohtosuka, T., Ito, H., Tanaka, A., 1997. Conversion of chlorophyll b to chlorophyll a and the assembly of chlorophyll with apoproteins by isolated chloroplasts. Plant Physiology 113, 137–147. Orthoefer, F., Dugan, L., 1973. The coupled oxidation of chlorophyll a with linoleic acid catalysed by lipoxidase. Journal of the Science of Food and Agriculture 24, 357–365. Perkins-Veazie, P., 1995. Growth and ripening of strawberry fruit. Horticultural Reviews 17, 267–297. Pichorner, H., Couperus, A., Korori, S., Eberman, R., 1992. Plant peroxidase has a thiol oxidase function. Phytochemistry 31, 3371–3376. Potty, V., 1969. Determination of protein in the presence of phenols and pectins. Analytical Biochemistry 29, 535–539. Rice-Evans, C., Miller, N., Paganga, G., 1997. Antioxidant properties of phenolic compounds. Trends in Plant Science 2, 152–159. Richard-Forget, F., Gauillard, F., Hughes, M., Jean-Marc, T., Boivin, P., Nicolas, J., 1990. Inhibition of horse bean and germinated barley lipoxigenases by some phenolic compounds. Journal of Food Science 60, 1325–1329. Sa´nchez-Romero, C., Garcı´a-Go´mez, L., Pliego-Alfaro, F., Heredia, A., 1995. One-step purification of an avocado peroxidase. Plant Physiology and Biochemistry 33, 531–537. Schuster, B., Herrmann, K., 1985. Hydroxybenzoic and hydroxycinnamic acid derivatives in soft fruits. Phytochemistry 24, 2761– 2764.
387
Shioi, Y., Masuda, T., Takamiya, K., Shimokawa, K., 1995. Breakdown of chlorophylls by soluble proteins extracted from leaves of Chenopodium album. Journal of Plant Physiology 145, 416–421. Shioi, Y., Tatsumi, Y., Shimokawa, K., 1991. Enzymatic degradation of chlorophyll in Chenopodium album. Plant Cell Physiology 32, 87– 93. Trebitsh, T., Goldschmidt, E., Riov, J., 1993. Ethylene induces de novo synthesis of chlorophyllase, a chlorophyll degrading enzyme in citrus fruit peel. Proceedings of the National Acadeny of Science USA 90, 9441–9445. Vicentini, F., Iten, F., Matile, P., 1995. Development of an assay for Mg-dechelatase of oilseed rape cotyledons, using chlorophyllin as the substrate. Physiologia Plantarum 94, 57–63. Watanabe, K., Ohta, H., Tsuchiya, T., Mikami, B., Masuda, T., Shioi, Y., Takamiya, K., 1999. Purification and some properties of pheophorbidase in Chenopodium album. Plant Cell Physiology 40, 104– 108. Yamauchi, N., Watada, A., 1994. Effectiveness of various phenolic compounds in degradation of chlorophyll by in vitro peroxidasehyrogen peroxide system. Journal of the Japanese Society for Horticultural Science 63, 439–444. Zieslin, N., Ben-Zaken, R., 1993. Peroxidase activity and presence of phenolic substances in peduncles of rose flowers. Plant Physiology and Biochemistry 31, 333–339.