Background

NADP (including the oxidized form NADP⁺ and the reduced form NADPH) is known as one of the most fundamental molecules for cellular metabolic reactions. In nature, NADP⁺ captures light-driven energy as electrons, via the photosynthetic electron transfer chain (PETC). Therefore, the level of available NADP⁺ in photosynthesis is quite important for autotrophic growth (Hashida and Kawai-Yamada, 2019).

Plant NAD kinase activity has been evaluated in many reports. According to in vitro assay on Arabidopsis thaliana tissue extracts, whole NAD kinase activity in 2-week-old seedlings was estimated as 560 nmol/h/mg protein (Turner et al., 2004). On the other hand, in 3-week-old rosette leaves of Col-0 it was calculated as 5.3 nmol/h/mg protein and 64 nmol/h/g FW (corresponding to approximately 12.1 nmol/h/mg protein) (Takahashi et al., 2006 and 2009). This large difference might be from variations in the amount of RubisCO (the most abundant leaf protein) between juvenile and mature leaves. Furthermore, it has been known that NAD kinase activity is under the control of various environmental stimuli, such as light, pathogen infection, oxidative stress etc. (Berrin et al., 2005). Beside transcriptional responses, several plant NAD kinases are known to be regulated via Ca²⁺ and calmodulin (CaM) signaling, cellular redox states and phytochromes (Tezuka and Yamamoto,1975; Delumeau et al., 2000; Hashida et al., 2018; Dell’ Aglio et al., 2019; Tai et al., 2019). Therefore, robust, reproducible, and standardized methods are indispensable for the evaluation of NAD kinase activity.

In Arabidopsis, the nadk2, a loss-of-function mutant in the chloroplastic NAD kinase (At1g21640) (Chai et al., 2005), lacked the light-responsive increase in NAD kinase activity (Hashida et al., 2018), suggesting that the activity of the NADK2 isoform is highly dependent on the light condition. Here we developed a novel NAD kinase assay, especially focusing on the chloroplastic NAD kinase activity. Compared to conventional in vitro NAD kinase assay, our newly developed method can drastically save time and costs, and it enables the evaluation of chloroplastic NAD kinase activity, while being comparable to the conventional method.