Ripening and in Vitro Retention of Respiratory Control by Avocado and Pear Mitochondria

Abstract

The retention of respiratory control ("surv-ival") The absence, or near absence, of ultrastructural change in the mitochondria of ripening pear (1), apple (17), and tomato (9) fruits plus the persistent capacity of mitochondria to oxidize substrates and demonstrate respiratory control throughout the climacteric of many fruits, including avocado (10, 14), tomato (8), apple (11-13), and banana (7) attest to the fact that mitochondria in senescing fruit cells remain functionally sound. Nonetheless, Romani et al. (26) discerned a ripening, age-related change in the mitochondrial system based on the observation that mitochondria in climacteric pears can neither withstand nor recover from doses of ionizing radiation that are tolerated by mitochondria in preclimacteric fruit.It has since been shown that isolated pear and avocado mitochondria can maintain RC' at 25 C for several hr (23,24). In analogy to experiments with long lived chloroplasts (6, 18), the maintenance of RC at 25 C has been referred to (24) as "survival." It seems reasonable to assume that survival is a more critical index of organelle integrity than a single point-in-time assessment of RC and should be helpful in understanding the status of mitochondria in ripening fruit. In the present study, the possible relationship between the capacity of mitochondria to survive in vitro and fruit ripening and senescence was investigated. 5 mM ,B-mercaptoethanol; 0.2%c PVP). The hard tissues of preclimacteric avocados were macerated by using a porcelain hand grater submerged in the isolation medium. A ratio of 1:3 (w/v), tissue to isolation medium, was used. After sedimentation at 14,000g the mitochondria were dispersed in wash medium (0.25 M sucrose; 50 mM K phosphate, pH 7.2; 0.1C% BSA; 5 mM 3-mercaptoethanol), centrifuged for 5 min at 1,200g to remove clumped particles and then at 8,000g for 10 min. The final pellets were dispersed in a few tenths ml of wash medium resulting in mitochondrial suspensions containing 20 to 30 mg protein /ml. Similar procedures were followed in the isolation of mitochondria from Bartlett pears with the exceptions, based on prior experiments (25), that the PVP concentration was increased from 0.2 to 0.5% (w,/v) and the pH during maceration was maintained within 6.7 to 7 by dropwise addition of I M KOH.Assay and Incubation. Oxidative activity of mitochondria at 25 C was measured with a Clark-type polarographic electrode. The sequence of additions into the cuvette was as follows: 3 ml of reaction medium, mitochondrial suspension, and repeated additions of ADP until anaerobiosis. The reaction mixture consisted of 0.25 M sucrose, 66 mm potassium phosphate buffer (pH 7.2), 1 mM MgCl,, 3.3 J.M CoA, 33 J.M thiamine pyrophosphate, 0.1 mM NAD, 1 mg/ml BSA, 100 j.g'ml chloramphenicol, and 10 mM substrate. Unless otherwise indicated, a-KG was supplied as substrate for both incubation and assay.For incubation at 25 C, done according to Romani and Ozelkok (24), 3 ml of reaction medium were placed in a 20-ml beaker 239 www.plantphysiol....

Mitochondrial Self-restoration as an Index to the Capacity of Avocado Fruit to Sustain Atmospheric Stress at Two Climacteric States

“…5). The apparent anomaly may be explained by the fact that mitochondria with higher RC and better RC maintenance are obtained from progressively riper avocados up to the climacteric peak (Ozelkok and Romani, 1975). Accordingly, both initial RC and RC maintenance by control mitochondria improved and was highest by the third extraction.…”

“…A quantitative estimate of RC, the respiratory control ratio (RCR) equals the respiratory rate in state 3 divided by that in state 4. These standard procedures are described in detail in Ozelkok and Romani (1975).…”

Inhibition of electron transport activities in mitochondria from avocado and pepper fruit by naturally occurring polyamines

“…Mitochondria in all cases were isolated by differential centrifugation (Ozelkok and Romani 1975) followed by purification on a continuous Percoll gradient according to the method of Moreau and Romani (1982) and used immediately. Mitchondria from avocados vary in density depending on the firmness of the fruit (Moreau and Romani 1975).…”