Increased Formation of Fluorescent Lipid- Peroxidation Products in Avocado Peels Precedes Other Signs of Ripening

Fluorescent products (lipofuscin-Iike compounds) of lipid peroxidation, which accumulate with age, were extracted from 'Fuerte' avocado (Persea americana Mill.) peels during ripening. Fractionation and analysis of these fluorescent compounds (FC’s) was carried out by an improved method, based on separation of FC’s from chlorophyll by Sep-Pak silica cartridges. A sharp rise in FC’s content was found 2 days after harvest in avocado fruits stored at 22 °C, and ethylene enhanced this rise 3-fold on the 4th day. The accumulation of FC’s preceded by at least 3 days the onset of climacteric ethylene and respiration and by 2 days the decrease in fruit firmness. Moreover, a 6-fold increase in the FC’s concentration occurred during 1 to 2 weeks of storage at 5 °C, but the avocado fruits did not show any other detectable signs of ripening. These results suggest that lipid peroxidation may be regarded as one of the earliest detectable processes occurring during fruit ripening. Thus, an increase of FC’s in peel may be employed as a horticultural characteristic for estimating initiation of ripening in avocado fruit. Biological membranes are labile to lipid peroxidation because of their high content of phospholipids and polyunsaturated fatty acids (PUFA). Several lines of evidence indicate that free radical-induced lipid peroxidation plays a role in bringing about many of the deteriorative changes associated with fruit ripening and leaf senescence (Galliard, 1968; Kimura et al., 1982; Thomas, 1986). In this respect, some suggested that fruit ripening may be viewed as an oxidative phenomenon, as indicated by results demonstrating that the onset of ripening correlates with the peroxide content of fruit tissues (Brennan and Frenkel, 1977; Brennan et al., 1979). Positive correlations between fruit senescence and oxidative or lipid degradation processes have been demonstrated during ripening of various fruits, such as avocado, pear, tomato (Brennan Received for publication 2 Oct. 1990. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, no. 3099-E, 1990 series. Supported by grant no. US-1525-88 from BARD, The United States Israel Binational Agricultural Research and Development Fund. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. Abbreviations: FC’s, fluorescent compounds. and Frenkel, 1977; Brennan et al., 1979) and litchi (Lin et al., 1988), and during aging of potato tubers (Lojkowska and Holubowska, 1989). The process of lipid peroxidation occurring during plant senescence yields, among other products, the formation of lipofuscin-like FC’s (Malshet and Tappel, 1973), via chain reactions consisting of PUFA, lipid hydroperoxides, and malondialdehyde (MDA) (Frankel, 1980; Harwood, 1988; Mudd, 1967). These fluorescent peroxidation products were shown to accumulate or increase with increasing age in various plant tissues and to be positively correlated with lipid peroxidation processes (Maguire and Haard, 1975, 1976; Meir et al., 1991; Pauls and Thompson, 1984; Wilhelm and Wilhelmova, 1981). Quantitative studies of these FC’s in climacteric fruits, such as banana and pears, employing a fluorimetric technique, showed an increase in their content during natural and ethylene-induced ripening (Maguire and Haard, 1975, 1976). However, the formation of FC’s has not been related to the pattern of changes in fruit ripening characteristics. Recently, we have developed a rapid and convenient method for extraction and determination of FC’s in green tissues. By employing this method we showed a remarkable increase in FC’s content of parsley leaves during natural and ethylene-induced senescence (Meir et al., 1991). Avocado is a climacteric fruit that does not ripen on the tree, but ethylene enhances its ripening after detachment (Biale, 1960; Eaks, 1966) when the fruit is stored at low (Zauberman and Fuchs, 1973) or high (Zauberman et al., 1988) temperatures. Since ripening is inhibited at low temperatures and lipid per oxidation processes still proceed, it was of interest to study the sequence of occurrence of these two events in avocado fruit. For this purpose, lipid peroxidation, exhibited by increased formation of fluorescent peroxidation products, was estimated by employing the method developed for determination of FC’s in green tissues (Meir et al., 1991). The pattern and time of onset of signs of ripening and changes in FC’s levels were studied in parallel during natural and ethylene-induced ripening of 'Fuerte' avocado fruit during storage at 5 and 22 °C. Materials and Methods PLANT MATERIALS AND TREATMENTS. Experiments were performed with 'Fuerte' avocado fruit harvested in midseason (October). Fruits were divided into three groups and stored from the day of harvest under the following conditions: a) Control fruits were stored at 22 °C and 85% relative humidity for 12 days until softening; b) ethylene-treated fruits were exposed to 100 μl ethylene/liter for 2 days in a chamber at 22 °C, and incubated subsequently in air at 22 °C for an additional 10 days; and c) fruits were stored at 5 °C for 14 days. Fruit firmness, respiration rate, ethylene evolution, and FC’s content were determined daily with samples of five fruits from each treatment. Where indicated, treatments were carried out in five replicates and the data presented are expressed as means ± standard error (SE). DETERMINATION OF FRUIT FIRMNESS. The firmness (Newtons required to penetrate the fruit) of five individual fruits was determined daily until the fruit was soft. We used a 'Chatillon' pressure tester, using a conical tip 6.5 mm in diameter (Fuchs et al., 1986; Zauberman et al., 1988). DETERMINATION OF RESPIRATION AND ETHYLENE. Five fruits from each treatment were sampled daily and their CO2 and ethylene evolution were determined by enclosing each fruit for l h in a 2-liter jar. The determinations of the gases were performed as described by Zauberman et al. (1988). The data obtained with a representative fruit are illustrated. EXTRACTION AND DETERMINATION OF PC’S. Fruits tested for firmness were used subsequently for determination of PC’s content. Lipofuscin-like fluorescent compounds were extracted from avocado fruit according to the procedure of Fletcher et al. (1973), as modified by Meir et al. (1991). Basically, 2-g samples of tissue, taken either from the peel or the pulp, were placed in a Polytron homogenizer (Kinematica, Luzern, Switzerland) with 20 ml chloroform-methanol (2:1, v/v) solution containing 20 mg butylated hydroxy toluene (BHT)/liter, homogenized for 15 sec, then warmed for 5 min at 45 °C. The homogenate was transferred to a centrifuge tube, mixed thoroughly with 10 ml 1.5 mM CaCl2 (Folch et al., 1957), and centrifuged (10 min, 10,000 x g). The chloroform-rich colored phase was processed further for removal of the interfering pigments: A 400-μl aliquot of the chloroform-rich layer [equivalent to 0.06 g fresh weight (FW)] was loaded onto a Sep-Pak silica cartridge (Waters Associates, Milford, Mass.), prewashed with 3 ml of analytical chloroform (Frutarom, stabilized with 0.7% ethanol). When the column was subsequently washed with 6 ml of analytical chloroform, the FC’s remained on the column, while the pigments (chlorophylls and carotenoids) were readily removed by the wash solution. The FC’s were then eluted with 2.5 ml methanol and the effluent was collected into tubes each containing 20 μl BHT (20 mg-ml methanol). The concentration of FC’s in these methanol-eluted fractions was determined by recording their fluorescence spectra and intensity (excitation at 300 and 355 nm, emission at 460 nm), using an Aminco spectrofluorimeter (model SPF-125). Standardization of the instrument was done with a solution of 1 μg quinine sulfate/liter (Sigma Israel Chemical Co.) dissolved in 0.1 M H2S04. Since all experiments were performed with equal quantities of tissue (0.06 g FW) and diluted to the same extent, one fluorescence unit was defined as the relative fluorescence measured. Thus, fluorescence intensity was expressed on the basis of units per 0.06 g FW. Chloroform extracts stored for 2 weeks at -20 °C gave reproducible results (±2%).