Nigericin

DCMU, DBMIB, ammonium chloride, and Antimycin A were purchased from Sigma–Aldrich and Nigericin was purchased from Nacalai Tesque Inc. To detect responsiveness to light, leaf discs were vacuum-infiltrated in the dark with a solution containing 10 or 100 µM DCMU, 10 or 100 µM DBMIB, 5 or 50 mM ammonium chloride, 10 mM DTT, 1 mM or 10 mM NaF and 50 µM Nigericin for at least 10 min before illumination. As a control, leaves were treated with 1% (v/v) ethanol (used as a solvent for the inhibitors). For isolated chloroplasts, equal volumes of solution containing 50 mM HEPES/KOH, 10 mM MgCl₂, 0.3 M sorbitol, and 10 mM NAD⁺ with 20 µM DCMU, 20 µM DBMIB, 10 mM ammonium chloride, or 10, 50, or 100 µM Antimycin A was added to the chloroplast suspension before illumination. Dark-response experiments were initiated immediately after the addition of an equal volume of solution containing 20 or 100 µM DCMU, 10 mM ammonium chloride, or 100 µM Antimycin A to 1% (v/v) ethanol in which the leaf discs were floating under light. Thereafter, leaf discs were incubated under either light or dark conditions.