A minute-scale P515 kinetic traces light-driven proton gradient formation in dark-incubated Synechocystis sp. PCC 6803.

The light-driven formation of a proton-motive force (pmf) across thylakoid membranes is crucial for ATP synthesis and photosynthesis in chloroplasts and cyanobacteria. Cyclic electron flow (CEF) around photosystem (PS) I is hypothesized to be a key contributor to pmf formation, but direct observation of CEF in vivo remains a major challenge. As one possible proxy, pmf formation can be measured on a millisecond scale using electrochromic shifts (ECS) of thylakoid pigments conventionally observed in plants through absorbance changes at a wavelength of 515 nm (P515). In this study, we describe a new P515 signal in the model cyanobacterium Synechocystis sp. PCC 6803, which can be observed on a time-scale of seconds to minutes upon red actinic light treatment. Treatments with uncouplers of electrochemical gradients and inhibitors of the photosynthetic electron transport chain indicate that the signal primarily traces proton gradient formation across the thylakoid membrane and suggest a major ECS contribution, but its precise origin remains to be deciphered. Still, the measuring routine allowed for phenotypic distinction between mutants with altered capacities for NDH- and PGR5-dependent CEF around PSI, thus enabling future research on both CEF pathways and photosynthetic trans-thylakoid pmf formation.