Effect of Electron Transfer Inhibitors and Uncoupling Agents on the Chlorophyll Fluorescence Lifetime during Slow Fluorescence Decline in Bean Leaves and Intact Chloroplasts

Abstract

Fluorescence measurements were performed with bean leaves and intact chloroplasts, which are characterized by a slow fluorescence decline. The fluorescence lifetime (ι) and the fluorescence relative quantum yield (φ) of intact chloroplasts, measured with the phase fluorometer, showed nearly proportional light-induced decrease with a halftime of ∼ 30 s, which was removed by 3-(3.4-dichlorophenyl)-1.1 -dimethylurea (DCMU), by 4.5,6,7-tetrachloro-2′-trifluoromethylbenzimidazole (TTFB). by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at a low concentration and by gramicidin D. Simultaneously the slow light-induced increase in absorbance at 518 nm (ΔA₅₁₈), reflecting thylakoid membrane energization, was eliminated. The data on ι of intact chloroplasts are original, the other results agree with literature data. In leaves, the slow light-induced fluorescence decline, with ι dropping from ∼ 2 to ∼ 0.6 ns, was abolished by FCCP at a concentration of 5 μm. However, while ι was stabilized at a level close to the initial (maximum) one or somewhat higher, φ became close to the minimum value. Besides, the amplitude of ΔA₅₁₈ was lowered about three times. These effects seemed to be due to multiple action of FCCP as a protonophoric uncoupler and an electron transfer inhibitor. In the presence of another uncoupling agent, TTFB, which, besides, is a diuron-like inhibitor of the electron transfer in chloroplasts, we observed the light-dependent, but hardly linked to changes in membrane potential, great increase in ι of a leaf from ∼ 1.9 to ∼ 4 .3 ns, with φ decreasing slightly. Addition of DCMU together with FCCP to the incubation medium or infiltration of a leaf with DCMU alone stimulated the rise in ι only to ∼ 3 ns. The increase in ι of a leaf observed in the presence of FCCP and DCMU, and especially with TTFB, may be associated with protein conformation changes which (i) alter the lifetime of nanosecond recombination luminescence of the photosystem II and/or (ii) disturb excitation energy transfer from the light-harvesting chlorophyll a/b complex to other pigment-protein complexes.