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Respiratory Metabolism of Pear Fruit and Cultured Cells Exposed to Hypoxic Atmospheres: Associated Change in Activities of Key Enzymes

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Respiratory Metabolism of Pear Fruit and Cultured Cells Exposed to Hypoxic Atmospheres: Associated Change in Activities of Key Enzymes J. AMER. SOC. HORT. SCI. 119(2):288–294. 1994. Respiratory Metabolism of Pear Fruit and Cultured Cells Exposed to Hypoxic Atmospheres: Associated Change in Activities of Key Enzymes George D. Nanos, Roger J. Romani, and Adel A. Kader1 Department of Pomology, University of California, Davis, CA 95616 Additional index words. Pyrus communis, controlled atmosphere, respiratory enzymes Abstract. ‘Bartlett’ pears (Pyrus communis L.) at two physiological stages, climacteric minimum or approaching the climacteric peak as achieved via storage for 2 or 8 weeks in air at 0C, respectively, were either ripened at 20C in air immediately or after exposure to 0.25% 02 for 4 days at 20C. Fruit stored for 2 weeks had relatively stable phosphofruc- tokinase (PFK), pyrophosphate : fru-6-P phosphotransferase (PFP), and pyruvate kinase (PK) activities but decreasing succinate dehydrogenase (SDH) activities during ripening in air. Similar fruit treated with 0.25% O2 had slightly increased PFK, PFP, and SDH activities and decreased PK activity. Fruit stored for 8 weeks exhibited higher levels of PFK and PFP activity upon transfer to 20C, in accordance with their more advanced physiological state. In general, the enzymic changes in these fruit upon exposure to 0.25% O2 and subsequent ripening in air were similar to those observed in the less-mature counterparts, most notable being an increase in mitochondrial SDH. Exposure of suspension-cultured pear fruit cells to hypoxia resulted in an accentuated rise in phosphoenolpyruvate carboxykinase activity and a dramatic rise in SDH activity upon transfer to air. Taken in concert, the enzymic analysis supports the hypothesis that the rise in succinate levels observed in hypoxic fruit tissues is the result of a partial reductive tricarboxylic acid cycle. Cytochrome oxidase activity did not change during hypoxia whereas soluble peroxidase decreased somewhat, perhaps a reflection of their Michaelis constants for O2. Hypoxia, or very limited O2 availability, is being tested as a Materials and Methods quarantine treatment and for possible long-term storage of fresh fruit and vegetables (Ke and Kader, 1992). Little information is Plant material and treatments. Mature-green ‘Bartlett’ pears available regarding the effects of hypoxia on the metabolism of were obtained on the day of harvest from Sacramento and Lake horticultural commodities. Ethanol is a known major end product counties, Calif. Fruit were stored at 0C until separate experiments of anoxic metabolism (fermentation), and it and acetaldehyde have were conducted on the pears from each area after 2 and 8 weeks of been shown to accumulate in hypoxic pears (Nanos et al., 1992). storage. Fruit were selected for uniformity of size and freedom Other probable anoxic end products include lactate, alanine, malate, from defects. Individual fruit were put in 450-ml jars and venti- α -aminobutyrate, and succinate (Davies, 1980). lated with air or 0.25% O2 (balance N2) using a continuous –1 The accumulation of succinate in anoxic fruit (Davies, 1980; flow-through system at 30 ml·min . After 4 days under 0.25% O2, Vanlerberghe et al., 1989) is commonly attributed to the suppres- the fruit were ventilated with air for 6 additional days. All atmo- sion of succinate dehydrogenase (SDH) (Butler, 1989). However, spheric treatments were conducted at 20C. Initially, and for every an alternative explanation for succinate accumulation suggests subsequent sampling period, three individual fruit replicates per that phosphoenolpyruvate (PEP) may be carboxylated via PEP treatment were evaluated. The sampling periods were 1, 2, 4, 6, and carboxylase (PEP-C) and possibly PEP carboxykinase (PEP-CK) 8 days during normal air treatment, 2 and 4 days for fruit under to produce oxaloacetate that, via partial reversal of the tricarboxy- 0.25% O2, and 1, 2, 4, and 6 days after their transfer to air. Changes lic acid (TCA) cycle, is in turn reduced to malate, fumarate, and in physiological state were assessed by measuring flesh firmness, finally succinate (Davies, 1980; Vanlerberghe et al., 1990). Inter- color change, and C2H4 and CO2 production. Enzyme assays mediates and some key enzymes in this sequence are shown in Fig. included those for phosphofructokinase (PFK), pyrophosphate : 1. In the search for underlying metabolic events, we investigated fru-6-P phosphotransferase (PFP), pyruvate kinase (PK), and the changes in the activity of these several regulatory enzymes in soluble PO. Mitochondria were isolated and SDH and CytOx ripening pears and fruit cells during and after their exposure to activities were measured. hypoxia. Suspension-cultured ‘Passe Crassane’ pear fruit cells were Since Nanos et al. (1992) found that preclimacteric pears seem grown as described by Pech and Romani (1979). At the end of their less stressed and to have greater potential for posthypoxia recovery log growth phase, the cells were transferred to a medium lacking than pears of a more-advanced physiological age, maturity was 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 days and then to incorporated as a variable. Cytochrome oxidase (CytOx) and aging medium, whereupon the atmospheric treatments were initi- peroxidase (PO) activities were also examined, as oxidase activity ated. Aging medium contained one-fourth the concentration of may be especially compromised during anoxia. Finally, nutrients found in growth medium and no 2,4-D, but was supple- suspension-cultured fruit cells were subjected to hypoxic stress mented with 0.4 M mannitol and 0.015 M sucrose. Two identical with subsequent enzymic analyses to assess if the cells could be experiments were conducted at 25C. The cell suspensions (three used to advantage in these types of studies. flask replicates per treatment) were treated atmospherically and periodically sampled exactly as the pears. Associated changes in Received for publication 28 Mar. 1993. Accepted for publication 6 Aug. 1993. The culture growth, cell vitality, and C2H4 and CO2 production have cost of publishing this paper was defrayed in part by the payment of page charges. been reported (Nanos et al., 1992). Under postal regulations, this paper therefore must be hereby marked advertise- ment solely to indicate this fact. Extraction and analysis of PFK and PFP. These two enzymes 1To whom reprint requests should be addressed. were extracted and assayed via a modification of the method of 288 J. AMER. SOC. HORT. SCI. 119(2):288–294. 1994. Fig. 1. Metabolic steps that may operate under hypoxia. Potential regulatory enzymes assayed in this study are shown. Smyth et al. (1984). Tissue from the equatorial area of the fruit, activity is reported as mmoles NADH oxidized/mg protein per including the skin, was ground to a fine powder under liquid N. min. Preliminary experiments were conducted to determine the Four grams of powder was extracted in 8 ml medium containing optimum extraction and assay pH (data not shown). M M M 100 m tris-HCl (pH 8.0), 2 m disodium-EDTA, 1 m MgCl2, 5 Isolation of mitochondria and analysis of SDH and CytOx. Pear mM dithiothreitol (DTT), and 200 mg insoluble (40,000 molecular fruit mitochondria were isolated using the method of Romani et al. weight) polyvinylpyrollidone (PVP). The brei was centrifuged at (1969). Fifty grams of peeled pear fruit tissue was macerated in 150 20,000× g for 25 min to remove debris, and the resulting superna- ml isolation medium. The isolation medium consisted of 0.25 M tant (crude homogenate) was used for enzyme assay. The reaction sucrose, 50 mM potassium phosphate (pH 7.2), 6 mM EDTA, 10 mM medium for PFK and PFP (total volume 2 ml) contained 90 mM β-mercaptoethanol, 0.5% soluble PVP, and bovine serum albumin M M –1 hepes-NaOH buffer (pH 8.0), 2.5 m MgCl2, 10 m fru-6-Pm, (BSA) at 1 mg·ml . The pH of the slurry was continuously 0.16 mM NADH, 1.2 units aldolase, 1.8 units glycerol-1-P dehy- adjusted to 7.2 with drop-wise additions of 5 N KOH. The homo- drogenase, 14 units triosephosphate isomerase, and 200 ml super- genate was squeezed through two layers of cheesecloth and centri- natant. The reaction was initiated by adding 1 mM sodium-ATP for fuged at 2000× g for 10 min. The supernatant was filtered through PFK activity or 1 mM sodium-pyrophosphate and 2 mM fruc- four layers of cheesecloth and centrifuged at 10,000× g for 15 min, tose-2,6 P2 for PFP activity. The absorbance at 340 nm was and the pellet was resuspended in 4 ml wash medium containing followed using a spectrophotometer at 25C (model PM2-DL; 0.25 M sucrose, 50 mM potassium phosphate (pH 7.2), and BSA at Zeiss, New York) and the rate was linear for at least 6 min. The 1 mg·ml–1. The resultant suspension was then centrifuged at 2000× activity is expressed in mmoles NADH oxidized/mg protein per g for 5 min, the resultant supernatant was centrifuged at 8000× g min for PFK and change in absorbance/mg protein per min for PFP. for 10 min, and the final pellet was resuspended in 0.5 ml wash The optimum extraction and assay pHs for these pear enzymes had medium to provide once-washed mitochondria. been determined (Kerbel, 1987). The method of Frenkel and Patterson (1973) with some modi- Extraction and analysis of PK. Five grams of tissue from the fications was used to assay for SDH. The reaction mixture con- equatorial area, including skin, was homogenized in 20 ml extrac- tained 100 mM potassium phosphate (pH 7.2), 10 mM KCN, 6 mM tion buffer [100 mM 2-(N-morpholino)-ethanesulfonic acid (MES), phenazine methosulfate, 20 mM succinate, and 0.2 mM pH 6.5, 5 mM DTT, and 0.5% soluble PVP] for 20 sec using a dichlorophenol indophenol.
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