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 Mg2+ in chloroplasts may contribute to the regulation of photosynthetic enzymes, but adequate methodology for the determination of free Mg2+ concentration ([Mg2+]) in chloroplasts has been lacking. We measured internal chloroplast [Mg2+] by using a Mg-sensitive fluorescent indicator, mag-fura-2. In intact, dark-kept spinach chloroplasts, internal [Mg2+] was estimated to be 0.50 mM, and illumination caused an increase in [Mg2+] to 2.0 mM in the stroma. The light-induced increase in [Mg2+] was inhibited by a blocker of driven electron transport and uncouplers. The K+-specific ionophore valinomycin inhibited the [Mg2+] increase in the absence of external K+, and addition of KCl restored the [Mg2+] increase. NH4Cl, which induces stromal alkalinization, enhanced the [Mg2+] increase. A Ca2+-channel blocker, ruthenium red, inhibited the [Mg2+] increase, but LaCl3 had no effect. These results indicate that stromal alkalinization is essential for light-induced increase in [Mg2+]. This system for measuring internal chloroplast [Mg2+] might provide a suitable system for assay of Mg2+ transport activity of chloroplast membranes.