Abstract
In this study, evidence is given that a number of isolated coupled plant mitochondria (from durum wheat, bread wheat, spelt, rye, barley, potato, and spinach) can take up externally added K(+) ions. This was observed by following mitochondrial swelling in isotonic KCl solutions and was confirmed by a novel method in which the membrane potential decrease due to externally added K(+) is measured fluorimetrically by using safranine. A detailed investigation of K(+) uptake by durum wheat mitochondria shows hyperbolic dependence on the ion concentration and specificity. K(+) uptake electrogenicity and the non-competitive inhibition due to either ATP or NADH are also shown. In the whole, the experimental findings reported in this paper demonstrate the existence of the mitochondrial K(+)(ATP) channel in plants (PmitoK(ATP)). Interestingly, Mg(2+) and glyburide, which can inhibit mammalian K(+) channel, have no effect on PmitoK(ATP). In the presence of the superoxide anion producing system (xanthine plus xanthine oxidase), PmitoK(ATP) activation was found. Moreover, an inverse relationship was found between channel activity and mitochondrial superoxide anion formation, as measured via epinephrine photometric assay. These findings strongly suggest that mitochondrial K(+) uptake could be involved in plant defense mechanism against oxidative stress due to reactive oxygen species generation.
Highlights
One of most outstanding problems in mitochondria bioenergetics concerns the mitochondrial permeability to metabolites, organic compounds, including vitamins, their derived cofactors, and metal ions
These findings strongly suggest that mitochondrial K؉ uptake could be involved in plant defense mechanism against oxidative stress due to reactive oxygen species generation
As far as plant mitochondria are concerned, even though mitochondrial structure and function are expected to be strictly dependent on Kϩ transport across the mitochondrial membrane, the knowledge of Kϩ permeability is not established at present
Summary
Chemicals and Plant Material—All reagents were purchased from Sigma. They were of purest available grade and they were used without further purification. Ofanto (250 g) were sowed on a distilled water-saturated polyurethane foam sheet, and they were covered with a Whatman filter paper. They were grown in the dark at 25 °C and 95% relative humidity for 72 h in an Heraeus HPS 1500 incubator. Purification of washed mitochondria was performed by isopycnic centrifugation in a self-generating density gradient containing 0.5 M sucrose, 10 mM Tris-HCl, pH 7.20, and 28% (v/v) Percoll (colloidal PVP-coated silica, Amersham Pharmacia Biotech) in combination with a linear gradient of 0% (top) to 10% (bottom) (w/v) PVP-40 (19).
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