Freezing Damage to Isolated Tomato Fruit Mitochondria as Modified by Cryoprotective Agents and Storage Temperature

Abstract

Isolated tomato (Lycopersicon esculentum var. Kc 146) fruit mitochondria could be stored successfully in the frozen state without a cryoprotective agent if the mitochondria were frozen quickly by immersion in liquid nitrogen and later thawed quickly at 30 C. Criteria of freezing damage were rate of respiration, adenosine diphosphate to oxygen ratio, and respiratory control ratio. Marked reduction in respiration and loss of respiratory control occurred when mitochondria were transferred from liquid nitrogen to -5, -10, or -18 C for 15 minutes prior to thawing at 30 C.Dimethylsulfoxide (5%Do) prevented freezing danmage when mitochondria were incubated at -5 C but did not prevent freezing damage at -10 or -18 C. There are a number of reports concerning freezing and animal mitochondria (8,(23)(24)(25) 33), but relatively little is known about how freezing affects isolated plant mitochondria. Heber and Santarius (18) reported that frozen and thawed spinach mitochondria partially retained phosphorylative and oxidative properties if sucrose was added, but protection was not as great as for photophosphorylation by chloroplasts. The spinach mitochondria were assayed with a-ketoglutarate as substrate, and all P/O ratios were less than 1. Hence, these workers may have observed the effect of freezing on the substrate level phosphorylation only.Our earlier work (5) showed that isolated tomato fruit mitochondria were severely damaged by freezing. Oxygen uptake decreased 89 /0 or more after 24 hr of storage at -18 C or in liquid nitrogen, and the damaged mitochondria lost respiratory control. The cryoprotective agent dimethylsulfoxide prevented freezing damage during storage in liquid nitrogen, and mitochondria were stored a month without decrease in ADP/O or loss of respiratory control.The present study also deals with freezing of isolated tomato mitochondria and is a continuation of the earlier work. Experiments were conducted to learn if DMSO3 was the only effective cryoprotective agent, whether rate of warming was related to a need for a cryoprotective agent, and the time course of freezing damage at several subzero temperatures.The cause of freezing damage is still not completely known (8,17,28), and the mode of action of cryoprotective agents is not clear (3,22,27,31). Isolated tomato mitochondria show promise for studying these phenomena. MATERIALS AND METHODSTomato fruit mitochondria were isolated and assayed with a Clark oxygen electrode as described earlier (5-7). Outer pericarp from mature green tomato fruits (Lycopersicon esculentunm, var.Kc 146) was ground at 0 C in a food mill with a medium (2 g of medium per g of tissue) containing 0.5 M mannitol, 0.05 Ni Na barbital (pH 7.8), 4 mm cysteine, 5 mm EDTA, and 1.5 mg/ml bovine serum albumin fraction V powder. The homogenate was strained through muslin, and mitochondria were collected as the fraction precipitating between 1,500g for 15 min and 15,000g for 15 min. Mitochondria were washed once in 0.5 M mannitol, 5 mm Na barbital, 1.5 mg,'ml bovine serum albumi...

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