Light-induced increase in free Mg2+ concentration in spinach chloroplasts: measurement of free Mg2+ by using a fluorescent probe and necessity of stromal alkalinization

Free Mg(2+) in chloroplasts may contribute to the regulation of photosynthetic enzymes, but adequate methodology for the determination of free Mg(2+) concentration (Mg(2+)]) in chloroplasts has been lacking. We measured internal chloroplast Mg(2+)] by using a Mg-sensitive fluorescent indicator, mag-fura-2. In intact, dark-kept spinach chloroplasts, internal Mg(2+)] was estimated to be 0.50 mM, and illumination caused an increase in Mg(2+)] to 2.0mM in the stroma. The light-induced increase in Mg(2+)] was inhibited by a blocker of driven electron transport and uncouplers. The K(+)-specific ionophore valinomycin inhibited the Mg(2+)] increase in the absence of external K(+), and addition of KCl restored the Mg(2+)] increase. NH(4)Cl, which induces stromal alkalinization, enhanced the Mg(2+)] increase. A Ca(2+)-channel blocker, ruthenium red, inhibited the Mg(2+)] increase, but LaCl(3) had no effect. These results indicate that stromal alkalinization is essential for light-induced increase in Mg(2+)]. This system for measuring internal chloroplast Mg(2+)] might provide a suitable system for assay of Mg(2+) transport activity of chloroplast membranes.