Phytochrome induces photoreversible calcium fluxes in a purified mitochondrial fraction from oats

Abstract

Previous studies have indicated that phytochrome regulates Ca2" fluxes across the plasma membrane of plant cells. In this study we investigated whether phytochrome can also regulate such fluxes across mitochondrial membranes, using the Ca2"-sensitive dye murexide to monitor the uptake and release of Ca2" by mitochondria. The results showed that Ca2" fluxes in these organelles could be photoreversibly altered, red light diminishing the net uptake rate and far-red light restoring this rate to its dark control level. Treatment ofthe mitochondria with ruthenium red blocked their Ca2+ uptake. In the presence of this inhibitor, red light induced a net efflux of Ca2`from the mitochondria, and subsequent far-red light reduced this efflux to nearly zero, the dark control level. Light-induced rate changes in Ca2`flux, both with and without the inhibitor, persisted for several minutes in the dark and remained photoreversible through several irradiations for as long as 30 min. The purity of the mitochondrial preparation wasjudged to be about 80% by electron microscopic morphometry; most of the phytochrome present was localized on the mitochondria in the preparation by using immunocytochemical methods. Taken together with previous findings, the results suggest that red light activation of phytochrome would initiate an increase in the cytosolic Ca2`concentration. The results are integrated with the fact that calmodulin is a component ofplant cell cytoplasms to construct a model postulating that phytochrome directs photomorphogenesis in part through its regulation of Ca2+ and calmodulin-controlled enzyme activities.

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

“…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). Roux et al (17) 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.…”

“…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).…”

“…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).…”