The Use of Fura-2 Fluorescence to Monitor the Movement of Free Calcium lons 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+ concentrations ( ~ 1 0 0 nM), 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.