Quantification of NAD(P)H in cyanobacterial cells by a phenol extraction method

Abstract

In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH 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 NADPH fraction [NADPH/(NADPH + NADP+)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction 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 NAD(P)H 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. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions.

Order-of-magnitude enhancement in photocurrent generation of Synechocystis sp. PCC 6803 by outer membrane deprivation

“…The amount of NADP + /NADPH was quantified using an NADP + /NADPH Assay Kit-WST (DOJINDO), following the manufacturer’s instruction. The extraction of intracellular NADP(H) of Synechocystis cells 81 was performed as follows: cells were collected centrifugally and resuspended in TE saturated phenol (NIPPON GENECO., LTD), followed by the addition of the same volume of Extraction Buffer supplied in the kit, vigorous mixing and centrifugation at 17,800 × g for 5 min. The obtained supernatant was transferred to a new tube and the same volume of chloroform was added.…”

Dark accumulation of downstream glycolytic intermediates confers robust initiation of photosynthesis in cyanobacteria

“…This allowed quenching and extraction of intracellular metabolites merely by directly dropping the cell suspension into the extraction solution during light irradiation, and avoided metabolic state variation during the sampling process. We chose phenol-chloroform-isoamyl alcohol (PCI) solution (pH 7.9) as an extraction solution because PCI extraction enabled rapid and efficient extraction of NADPH from Synechocystis (26). Note that the exchange of medium for NaHCO 3 solution is a typical pretreatment for measuring photosynthetic activity of fresh-water cyanobacteria (27).…”

“…Carefully dropping the cell suspension into the extraction solution during light irradiation enables the rapid quenching and extraction of intracellular metabolites and avoids metabolic state variations during sampling. A phenol-chloroform-isoamyl alcohol (PCI) solution (pH 7.9) is selected as the extraction solution because PCI extraction enables rapid and efficient extraction of NADPH from Synechocystis 23 . Second, stable isotopomers of the analyte compounds are used as internal standards (IS).…”

Respiration Interacts With Photosynthesis Through the Acceptor Side of Photosystem I, Reflected in the Dark-to-Light Induction Kinetics of Chlorophyll Fluorescence in the Cyanobacterium Synechocystis sp. PCC 6803

“…We used two cyanobacterial strains, in which the redox states of NADPH are different but those of the PQ pool are similar after dark-acclimation, in order to verify the effect of NADPH on chlorophyll fluorescence. One of the two strains is Δ ndhF1 , in which the PQ pool is oxidized ( Ogawa et al, 2013 ) while NADPH is reduced in the dark ( Mi et al, 2000 ; Tanaka et al, 2021 ). The other is the strain with disrupted gnd gene (Δ gnd ), which encodes 6-phosphogluconate dehydrogenase ( Kaneko et al, 1996 ) in oxidative pentose phosphate (OPP) pathway, the major pathway producing NADPH in the dark ( Cheung and Gibbs, 1966 ; Wolk, 1973 ).…”