THE USE OF FURA-2 FLUORESCENCE TO MONITOR THE MOVEMENT OF FREE CALCIUM-IONS INTO THE MATRIX OF PLANT-MITOCHONDRIA (PISUM- SATIVUM AND HELIANTHUS-TUBEROSUS)

Purified mitochondria isolated from pea (Pisum sativum 1. cv Alaska) stems and Jerusalem artichoke (Helianthus tuberosus 1. cv 061) tubers were loaded with the acetoxymethyl ester of the fluorescent Caz+ indicator fura-2. This made possible the continuous monitoring of free [Ca”] in the matrix ([Ca”],) without affecting the apparent viability of the mitochondria. Pea stem mitochondria contained an initial [Ca2+], of approximately 60 to 100 nM, whereas [Caz+], was severalfold higher (400-600 nM) in mitochondria of Jerusalem artichoke tubers. At low extramitochondrial Ca2+c oncentrations ( ~ 1 0 0nM ), there was an energy-dependent membrane potential increase in [Ca’+],; the final [Caz+], was phosphate-dependent in Jerusalem artichoke but was phosphateindependent in pea stem mitochondria. The data presented indicate that (a) there is no absolute requirement for phosphate in Caz+ uptake; (b) plant mitochondria can accumulate externa1 free Caz+ by means of an eledrophoretic Caz+ uniporter with an apparent affinity for CaZ+ (K, approximately 150 nM) that is severalfold lower than that measured by conventional methods (isotopes and Ca2+-sensitive electrodes); and (c) [Ca”], is within the regulatory range of mammalian intramitochondrial dehydrogenases.