Succinoxidase Activity of Avocado Fruit Mitochondria in Relation to Temperature and Chilling Injury throughout the Climacteric Cycle

Abstract

Mitochondrina were isolated from 'Fuerte' avocado fruit (Persea americana Mill.) at four different stages of the respiratory climacteric. PrecUmacteric fruit had the highest rate of succinate oxidation and the postdimacteric mitochondria the lowest. Subsequently, successive additions of ADP increased the respiratory control ratio. Arrhenius plots of succinate oxidation of intact mitochondra from dimacteric rise and climacteric peak fruit showed two transition temperatures , while only one was observed in predlimacteric fruit. The low tempertore phase transition was at about 9 C, while the high one was at 20 C. In postclimacteric fruit, the low temperature transition decreased to between 5 and 2 C. The state 3 rate of succinate oxidation was highest for mitochondria from preclimacteric fruit and decreased for each later stage. The state 4 rates for preclimacteric and climacteric rise were the same, while both the climacteric peak and postclmacteric rates were about 40% lower than the predimacteric 02 uptake. The results indicate continuous changes in the mitochondrial membrane of the electron transport chain throughout the climacteric cycle. The change in the membrane influencing the phosphorylation system is greatest between cimacteric rise and peak stages. Mitochondrial membranes of postdmacteric fruit are presumed to change from flexible disordered to solid ordered phase at a lower temperature than those of other climacteric stages. The changes occurring in ripening which govern the initiation of the respiratory climacteric have not been identified. A number of proposed mechanisms have been reviewed (21). Mitochondria have been examined from several fruit including avocado (9), apple (5), and tomato (3), at various stages of the climacteric, and shown to: (a) oxidize tricarboxylic acid cycle acids at all stages; (b) exhibit respiratory control; and (c) phosphorylate efficiently at all stages of ripening. Electron micrographs show no change in the organelle morphology between preclimacteric and postclimacteric stages of avocado fruit (W. Vanderwoude and R. E. Young, personal communication). We observed recently that postclimacteric avocados were less sensitive to chilling injury than at any other stage (8). The chilling sensitivity temperature has been shown by Lyons and Raison (12) to be correlated with the temperature at which an abrupt change occurs in the activation energy of succinoxidase activity in isolated mitochondria, and this change is believed to I reflect a change in the lipid fraction of the mitochondrial membrane from a fluid to a solid phase. The change of lipid in the membrane from liquid to solid increases the activation energy of the enzyme complex by causing a conformational alteration of the membrane-bound enzyme. The change in chilling sensitivity suggested to us that ripening may be regulated by membrane lipids rather than by changes in the enzyme proteins themselves. We report here studies of Arrhenius plots of succinoxidase activity for avocado mitochon-dria at four stages of the respiratory climacteric as evidence for changes in the lipid components of the mitochondrial membrane. MATERIALS AND METHODS Mature 'Fuerte' avocado fruit (Persea americana Mill.) of uniform size were harvested from one tree at the South Coast Field Station of the University of California at Irvine. Fruit were placed in individual 2-liter wide mouth jars at 20 C. CO2 production was monitored by Beckman model 215 IR gas analyzer as described previously (7). As soon as a fruit achieved a respiratory rate of a particular stage on the climacteric, the fruit was cooled to 2 to 4 C prior to cutting and grating. Mitochondria were isolated and immediately assayed for succinoxidase activity as described below. Isolation Procedure. The technique used throughout this study was that described by Lance et al. (9) with modifications suggested by Romani and Ozelkok (22) and by Laties and Treffry (10). Each fruit was peeled, the embryo and seed coat removed, and the fruit cut into about 10 12-g pieces. These were mixed with 350 ml of isolation medium and poured into a stainless steel Oster automatic juice extractor lined with two layers of Miracloth. In the case of postclimacteric fruit, the homogenate would often not pass through Miracloth and four layers of cheesecloth were substituted. The homogenate was adjusted to pH 7.4. The isolation medium consisted of 0.35 M sucrose, 50 mM tris buffer (pH 7.9), 5 mm EDTA, 2 mM MgCl2, 6 mm KCl, 5 mm cysteine-HCl, 0.2% POP (40,000 mol wt), and 0.1% BSA. Cellular debris was removed by centrifu-gation at 1,600g for 8 min. After an additional centrifugation of the supernatant at 16,000g for 12 min, the mitochondria pellet was resuspended in 250 ml "wash" medium composed of 0.3 M sucrose, 10 mm K-phosphate buffer (pH 7.2), 25 mm tris buffer (pH 7.2), 1 mM MgC12, 0.1% BSA, and 5 mM /-mercaptoethanol. The suspension was centrifuged at 1,000g for 5 min and the resultant supernatant fraction at 8,000g for 10 min. The final pellet was resuspended with a Teflon and glass Potter homogenizer in 1.3 ml wash medium containing 5 ,umol of ATP. The total volume was approximately 2 ml containing 20.8 to 32.5 mg protein. Succinoxidase Assay. Mitochondrial suspension was assayed 470