Calcium Accumulation by Maize Mitochondria

Abstract

A number of laboratories have reported that animal mitochondria can actively accumulate various inorganic ions (8). Energy can be supplied by either substrate oxidation or ATP. The antibiotic, oligomycin, inhibits ATP-driven Ca uptake, but not substrate-driven uptake (6,18 Materials and MethodsIsolation of Mitochondria. Corn seeds (Zea mays L., WF9 X M14) were germinated in the dark at 28°on paper towels saturated with 10-4 M CaC2.After 3 and one-half days, the shoots were excised, chilled, and ground in an ice-cold mortar with 0.25 M sucrose, 0.05 M KH2PO4 and 0.005 M EDTA, adjusted to pH 7.5 with Tris (hydroxymethyl) aminomethane. The slurry was strained through cheesecloth. Mitochondria were isolated in a refrigerated centrifuge as the fraction sedimenting between 2000 X g for 5 minutes and 12,000 X g for 10 minutes. The mitochondria were twice washed, first in the grinding medium, next in 0.25 M sucrose and were finally suspended in 0.25 M sucrose. The mitochondria were quite active, giving QO2(N) values of about 1500 and P/O values of about 2.5 when oxidizing a mixture of pyruvate and succinate in the absence of inhibitors or uncouplers. Procedures for determining oxidative phosphorylation have been described (12

Stimulation of Mitochondrial Calcium Uptake by Light during Growth of Corn Shoots

“…For.the accumulation of Ca2' by plant mitochondria, energy can be supplied by either substrate oxidation or exogenously added ATP (7,8). Oligomycin specifically inhibits the ATPdriven Ca2+ uptake, but not the substrate-dependent uptake, ishes the net uptake rate, following the induction of Ca2" efflux from mitochondria, and far-red light restores this rate to its dark control level when both succinate and ATP are used as the energy source.…”

“…For.the accumulation of Ca2' by plant mitochondria, energy can be supplied by either substrate oxidation or exogenously added ATP (7,8). Oligomycin specifically inhibits the ATPdriven Ca2+ uptake, but not the substrate-dependent uptake, suggesting that a high energy intermediate of oxidative phosphorylation is the direct energy source for mitochondrial Ca2+ uptake in plants (8) and in animals (9).…”

“…The cytoplasmic concentration of free Ca2" is thought to be in the range of 10-8 to 10-5 M (1, 18) and this low Ca2" concentration is maintained against high Ca2" concentrations outside the cells and inside the various membrane systems. The regulation for maintaining the low cytoplasmic Ca2" is thought to involve both the accumulation of Ca2+ in subcellular organelles, in particular mitochondria, through an active Ca2+ transport system (8,17) and an active extrusion through the plasma membrane (4).For.the accumulation of Ca2' by plant mitochondria, energy can be supplied by either substrate oxidation or exogenously added ATP (7,8). Oligomycin specifically inhibits the ATPdriven Ca2+ uptake, but not the substrate-dependent uptake, ishes the net uptake rate, following the induction of Ca2" efflux from mitochondria, and far-red light restores this rate to its dark control level when both succinate and ATP are used as the energy source.…”

“…Dieter and Marme (3) also suggested the regulation of mitochondrial Ca2" fluxes by phytochrome; however, in their system the active accumulation of Ca2" into the mitochondrial fraction from etiolated corn coleoptiles is inhibited when the seedlings are irradiated with far-red light. In any case, the properties of the active Ca2" fluxes by plant mitochondria seem to be altered by light, through the action of phytochrome.Most Ca2" flux assays have been performed with mitochondria isolated from the etiolated plant tissues (4,8,17), because of the difficulty in isolating the preparation free from other organelles from the green tissues. However, the Percoll discontinuous density gradient method permits the isolation of pure and metabolically competent mitochondria from leaves (14).…”

“…The cytoplasmic concentration of free Ca2" is thought to be in the range of 10-8 to 10-5 M (1, 18) and this low Ca2" concentration is maintained against high Ca2" concentrations outside the cells and inside the various membrane systems. The regulation for maintaining the low cytoplasmic Ca2" is thought to involve both the accumulation of Ca2+ in subcellular organelles, in particular mitochondria, through an active Ca2+ transport system (8,17) and an active extrusion through the plasma membrane (4).…”