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

Kenya Tanaka,Shuji Nakanishi,Ginga Shimakawa,Hiro Tabata,Chikahiro Miyake,Shoko Kusama

doi:10.1007/s11120-021-00835-1

Kenya Tanaka, Shuji Nakanishi + Show 4 more

Open Access

https://doi.org/10.1007/s11120-021-00835-1

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Journal: Photosynthesis Research	Publication Date: May 1, 2021
Citations: 15	License type: open-access

Affiliation: Osaka University, Kobe University

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.

Highlights

The redox pair N ADP+/NADPH is involved in various reactions in photosynthetic organisms. NADP+ is the terminal electron acceptor in the photosynthetic electron transport chain (PETC) and is converted to NADPH under light conditions
To verify the possibility that NADPH was unexpectedly oxidized during the extraction process, extraction was performed using a buffer containing exogenous 1.5 μM N ADP+ and 1.5 μM NADPH as standards
It was confirmed that phenol / chloroform / isoamyl alcohol (PCI) itself did not affect the measurement of exogenous NADP(H) (Fig. 1b–3) These results suggested that the intracellular concentrations of NADP(H) can be quantitatively determined using the developed PCI extraction protocol

Summary

Introduction

The redox pair N ADP+/NADPH is involved in various reactions in photosynthetic organisms. NADP+ is the terminal electron acceptor in the photosynthetic electron transport chain (PETC) and is converted to NADPH under light conditions. The redox pair N ADP+/NADPH is involved in various reactions in photosynthetic organisms. NADP+ is the terminal electron acceptor in the photosynthetic electron transport chain (PETC) and is converted to NADPH under light conditions. Given that the N ADP+/NADPH redox ratio, a critical factor influencing various biological processes, can vary with changes in the lighting conditions, it is vitally important to quantitatively determine the intracellular concentrations of NADP(H) for a deeper understanding of the physiology of photosynthesis. Fluorescence detection of NADPH is a representative in vivo method for measuring light-responsive changes in NADPH concentrations. The fluorescent yield of NADPH changes depending on the peripheral environment (Latouche et al 2000; Kauny and Sétif 2014). NADP+ (the counterpart of NADPH) does not emit fluorescence. The in vivo fluorescence-based technique cannot directly assess the NADP+/

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