One millisecond delayed light emission has been studied in isolated coupled lettuce (Lactuca sativa var. romaine) chloroplasts. Delayed light emission was increased upon addition of ferricyanide or 2,3, 5,6-tetramethyl-p-phenylene diamine. In the presence of ferricyanide, the magnitude of the signal was increased by the addition of ADP (in the absence of orthophosphate), ATP, DIO-9, or phlorizin. The signal was also increased by the addition of NaCI and by the addition of NH4Cl in the presence of a high NaCl concentration. The signal of delayed light emission was decreased by the addition of gramicidin, valinomycin, and by the addition of NH4Cl in the presence of a low NaCl concentration.Phosphorylation, whether started by addition of ADP or by addition of glucose plus hexokinase plus ATP, caused a significant decrease in delayed light emission. It was concluded that the magnitude of delayed light emission reflects the size of the proton motive force across the thylakoid membrane. Calibration of delayed light emission by creating KCI gradients indicated that the value for the electrochemical potential gradient for H+ in the presence of ferricyanide was at least 155 millivolts decreasing to 134 millivolts after the onset of phosphorylation.Goedheer (8) has suggested that DLE' in photosynthetic organisms results from back reactions of electrons and holes in acceptor and donor pools of photosystem II or bacterial reaction centers in a reversal of the light reactions. On the basis of studies involving the effect of uncouplers and phosphorylating agents on DLE in chloroplasts, Mayne (17) suggested that the high energy state of phosphorylation is an additional source of energy for DLE, in direct equilibrium with the photochemical act. Subsequently, Fleischmann (7) suggested that during the photoact charge separation across the thylakoid membrane would provide a store of energy in the form of electrical potential. This so-called membrane potential provides a source of energy in the dark for DLE by presumably lowering the activa- tion energy for the recombination process. According to a more detailed model (6), the free energy difference between donor and acceptor pools is dependent (in addition to the concentration of Z+ and Q-) both on the proton concentration difference (ApH) and the electrostatic potential difference (AE) across the thylakoid membrane. Since according to the chemiosmotic hypothesis, AE and ApH comprise the driving force for ATP formation (19), DLE should indeed be closely related to photophosphorylation. In the present work, we have further studied this relationship using coupled phosphorylating lettuce chloroplasts. In addition, following the method of Barber (4, 5), the magnitude of the minimal value of the p.m.f. was estimated during electron flow and when electron flow was coupled to phosphorylation.
MATERIALS AND METHODSChloroplasts were isolated from lettuce leaves Lactuica satii'a var. romaine. One hundred grams of leaves were homogenized for 15 sec in a Waring Blendor in 130 ml of...
