Isolation of Active Mitochondria From Tomato Fruit

Abstract

Abstract. An improved method for isolating mitochondria from tomato fruit (Lycopersicon esculentum Mill.) is described. The fruit is chilled, and the tissue of the fruit wall cut by hand into very thin slices with a razor blade while immersed in a buffer containing 0.4 MI sucrose, 2 mm MgCl9, 8 mm EDTA, 4 mm cysteine, 10 mm KC1, 0.5 mg per ml bovine serum albumin 50 mm tris-HCI, pH 7.6. The pH is monitored and kept within the range of 7.0 to 7.2 by dropwise addition of 1 N KOH during cutting. The tissue is strained through 8 lavers of cheesecloth and centrifuged at 2000 X g for 15 minutes. The supernatant is then centrifuged at 11,000 X g for 20 minutess and the sediment is washed onoe with a medium oontaining 0.4 M sucrose, 10 mm KCI, 1 mm MgCl,,, 10 mm tris-HCl, 10 mmi KH9PO4 and bovine serum albumin (0.5 mg per ml), pH 7.2. Electron microscope studies show that this method gives homogeneous, relatively intaot mitochondria; they have a higher respiratory control ratio than those reported by other workers. The method was also tested successfully on fruits of cantaloupe and 'Honey Dew' melon.For further studies of fruit ripening and the role of ethylene, preparations of isolated mitochondria were desired which would have a high degree of respiratory control by ADP, good ADP/O values in accord with the generallv accepted limiting ratio, and a high degree of homogeneity with close resemblance between the fine structure of the isolated mitochondria and those in intact tissue sections. Various techniques for isolating mitochondria from fruits have been reported (3,7,12,14,21 sequent steps were carried out at this temperature. The chilled fruits were cut into 2 or more pieces, and the seeds and the highly acid gelatinous placental material were carefullv and completely removed. Portions of the outer and radial wall tissue (60 g) were immediately washed with buffer and immersed in buffer, where they were cut by hand into very thin slices with a stainless steel razor blade (fig 1). The buffer was based on one used by Lance et al. (14), consisting of 0.4 M sucrose, 4 mM cvsteine, 2 mm MgCl,, 10 mm KCl, 8 mm EDTA, 50 mM tris-HICl, and 0.5 mg per ml of BSA (bovine serum albumin, Fraction V, unesterified fatty acid poor) at pH 7.6. A pH range of 7.0 to 7.2 was maintained throughouit isolation with dropwise additions of 1 N KOH. The suspension was strained through 8 layers of cheesecloth, and the isolation procedure was completed as summarized in figure 1;

Oxidative Activity of Mitochondria Isolated from Plant Tissues Sensitive and Resistant to Chilling Injury

“…Thus, where plant material is given a differential pretreatment, it is most difficult to ascertain whether the observed differences in activity represent a direct effect of the chilling treatment on the mitochondria or an indirect effect due to the release or activation of some components which might be operational during isolation. It has also been noted that the method of tissue disruption (8), the amount of mitochondrial protein in the assay mixture (20), and the time involved between tissue disruption and assay (18) influence the activity of isolated mitochondria but which may be of little significance in vivo.…”

Studies on the Respiratory Properties of Mitochondria Isolated from Developing Winter Wheat Seedlings

“…Materials were introduced into the chamber through a capillary by a syringe, and the reaction mixture was stirred by means of a magnetic stirrer. The reaction mixture was composed of 0.30 M mannitol, 10 (15), and by microkjeldahl (26).…”

Active Mitochondria Do Not Produce Ethylene

“…We have been able to isolate very acti%ve, homogeneous. relatively uininjured mitochondria fronm various fruits (6). It is the purpose of this article to indicate that such active mitochondria do not directly produce ethvlene; it appears more likely that ethylene is l)roduced by a soluble enzyme.…”

“…VC 243-20) were harvested at various stages of maturity from greenhouse-grown plants (12). The fruits were washed with distilled water and chilled (0-40) from 1 to 4 hours before processing by the method of Ku et al (6). The flesh of the fruit wall was carefully removed, avoiding the inclusion of any skin, seeds, or the jellv-like tissue around the seeds.…”

“…Electron micrographs show that fraction I contains nuclei, grana, plastids, small vesicles, mitochondria, and unidentified organelles and fragments, while the mitochondrial fraction is a homogeneous preparation of relatively uninjured mitochondria (6). Fraction I and the mitochondrial fraction were each suspended in a known volunme of buffer, containing 0.25 M sucrose, 10 mm KH.,PO4, 0.5 mg per ml BSA, 10 mm KCI, pH 7.4. and dialyzed against the same buffer for 2 hours, changing the buffer once.…”