Diphenyl carbazide restores electron transport in isolated, illuminated chloroplasts after electron transport from water has been eliminated by mild heat treatment

Abstract

Freshly isolated, illuminated chloroplasts oxidize water and transfer the resulting electrons through the photosynthetic electron transport chains in their thylakoid membranes to the artificial electron acceptor, dichlorophenol indophenol (DCPIP). As a consequence, DCPIP is reduced and the decline in absorbance over time can be used to measure the rate of electron transfer. When gently heated, chloroplasts lose the capacity to oxidize water and the transfer of electrons to DCPIP is eliminated. Electron transport through chloroplasts to DCPIP is restored in the presence of the artificial electron donor diphenylcarbazide (DPC). If students gain experience with the DCPIP photoreduction assay and are given information on normal chloroplast function, they should be able to predict the behavior of heat-treated chloroplasts in a variety of experimental conditions. A number of such predictions are outlined and tested. The experiments can all be conducted with a limited repertoire of equipment and easily prepared solutions. Consequently, this work is well suited to an investigative study in which each student group, in consultation with instructors, can make and test its own prediction. The ways in which changing different variables can affect the quality of the experimental results is emphasized. Additional studies, on measurements of rates of oxygen evolution and emitted chlorophyll fluorescence, are briefly described to support the inferences that heat-treated chloroplasts do not oxidize water and that the vectorial transfer of electrons through them to DCPIP is identical to that in untreated chloroplasts.