A quantitative demonstration of NADP+/NADPH redox homeostasis in cyanobacterial cells

In photosynthetic organisms, it is recognized that the intracellular NADP+/NADPH ratio is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADP+/NADPH ratio quantitatively estimated to date. In the present study, the light-response of the NADP+/NADPH ratio was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light-response of NADP(H) observed using PCI extraction was qualitatively consistent with the NADPH fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light-response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light-dark transition, indicating that some NADP(H) is not light-responsive. Specifically, 64% of total NADP(H) was observed as non-light-responsive in the wild-type cells. The variation of the intracellular NADP+/NADPH ratio is limited to a narrow range due to the presence of non-light-responsive NADP(H).