The Effect of Inhibitors and Uncouplers of Photosynthetic Phosphorylation on the Delayed Light Emission of Chloroplasts*

Abstract

Measurements were made of the 3.7 msec delayed light emission of chloroplasts treated with a variety of agents which affect the rate of electron transport (Hill reaction) or photosynthetic phosphorylation. The presence of the electron acceptors ferricyanide or pyocyanine increased delayed light emission. Inhibitors of electron transport (3-(3,4dichloro-pheny1)-1, -I-dimethylurea or l,lO(ortho)-penanthroline) inhibited delayed light emission. Uncouplers of photosynthetic phosphorylation caused a decrease in delayed light emission. The addition of a phosphate acceptor system inhibited delayed light emission. This inhibition was reversed by inhibitors of the phosphorylation reaction, e.g. Dio-9 or phlorizin. From these results it was concluded that the 3.7 msec delayed light emission probably occurs as a result of back reactions of intermediates in the coupled electron transport and photosynthetic phosphorylation systems. I N T R O D U C T I O N IN 1951 Strehler and Arnold(') observed a delayed luminescence of an alga. This luminescence had a spectrum corresponding to the fluorescence of chlorophyll in v~v o , (~) indicating the formation in the dark of the first excited singlet state via some back reaction.To explain the phenomenon, Arnold and Sherwood(s) proposed a semiconductor type mechanism in which the luminescence resulted from a recombination of electrons and holes within the grana of the chloroplasts, which they had shown to have semiconductor-like properties. Strehler,(4) on the other hand, preferred a mechanism based on the backreactions of discrete chemical intermediates formed in the light.Albrecht et d,@) Goedheer@) and Bertsch(') have demonstrated the interaction of the two photosystems(*) of green plants in the phenomenon of delayed light emission (DLE).? Bertsch and Bishop(Q) have shown that a Scenedesmus mutant which lacks photosystem I1 has a greatly decreased DLE, while a mutant which lacks photosystem I has an increase in the rapid DLE. Clayton and Bertschc'O) reported that a mutant Rhodopseudomoms spheuoides, which lacks reaction centers and is unable to perform any of the usual photochemistry, has its steady state DLE reduced at least 300 fold compared to the wild type, indicating the necessity of an intact photosystem for normal DLE.The objective of this paper is to present some experimental results which relate the rapid (3.7 msec) delayed light emission to back reactions of electron transport components coupled to the phosphorylation system. *Contribution No. 256 of the Charles F. Kettering Research Laboratory, Yellow Springs, Ohio, U.S.A. t Abbreviations : DLE-delayedlight emission; DCMU-3-(3,4-dichlorophenyl)-lt -1-dimethylurea; CCCP-189 m-chlorocarbonyl cyanide phenylhydrazone; tricine-N-Tris (hydroxymethy1)-methyglycine.

The Relation between Photophosphorylation and Delayed Light Emission in Chloroplasts

“…It has been reported previously that in the presence of phosphorylating reagents the extent of DLE is diminished (17 (Fig. 7).…”

“…Several uncouplers were shown previously to inhibit DLE (17). Upon the addition of gramicidin, we have observed that the extent of "discharge" of the signal by subsequent addition of ADP decreased with increasing concentrations of the uncoupler.…”

Berger C. Mayne (1920–2011): a friend and his contributions to photosynthesis research

“…Mayne and Clayton (1967) examined the effects of a variety of electron transport and phosphorylation inhibitors and phosphorylation uncouplers on DLE and found that, under a variety of conditions, the intensity of DLE mirrored the predicted magnitude of the so-called high-energy phosphorylation intermediate. DLE increased when Hill reaction electron acceptors were added, and was inhibited by PSII inhibitors such as DCMU [3-(3,4-dichlorophenyl) 1,1 dimethylurea] and by phosphorylation uncouplers.…”

“…This ''proton gradient'' would be the concentration part of the protonmotive force (pmf) postulated to be the ''high energy intermediate'' in Peter Mitchell's chemiosmotic hypothesis (Mitchell 1961). Mayne and Clayton (1967) reasoned that if the high energy intermediate were the precursor of delayed fluorescence, and if it could be generated by an acid-base transition, it should be possible to produce light emission by an acid-base transition-in effect a reversal of the light-driven formation of the proton gradient. They subjected chloroplasts to a similar acid-base transition in front of a photomultiplier, and found that a burst of light was indeed emitted when the base was injected (Mayne 1966;Mayne and Clayton 1966).…”

Estimation of the activation energy for millisecond delayed fluorescence from uncoupled chloroplasts

“…It has become clear from the work of Mayne [2,3] and subsequent workers [4,5] that the high-energy state of phosphorylation acts on this back reaction. This effect is most readily observed in the 1 msec region of the decay.…”