Lots of regulators have been found playing important roles in regulating NADK activity. According to whether Ca²⁺/CaM is needed for NADK activation, NADKs can be divided into two types in plants, Ca²⁺/CaM dependent NADKs and Ca²⁺/CaM independent NADKs. Simon et al. (1984) found that in chloroplasts of pea seedlings, a Ca²⁺/CaM dependent NADK exists in the envelope of chloroplasts whereas a Ca²⁺/CaM independent NADK is found in the stroma of the organelle. Ca²⁺ and CaM are necessary for the Ca²⁺/CaM dependent NADKs. Roberts et al. (1986) reported that lycine-115 methylated CaMs activated an NAD kinase preparation to a maximal level that was at least 3–4-fold lower than the level of activation obtained with the corresponding unmethylated CaMs. Two tobacco transgenic plants, VU-1, which expressed a CaM that can be methylated at the lycine-115 residue, and VU-3, which expressed a CaM where the lycine-115 was substituted with arginine and was incapable of methylation, exhibited different growth behaviors (Roberts et al., 1992). VU-1 showed no difference compared to wild type, but VU-3 lines were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. A possible reason is that overexpressing VU-3 in plants led to an alteration in cellular metabolism due to the disruption of nicotinamide co-enzyme homeostasis (Roberts et al., 1992). Harding et al. (1997) found that VU-3 lines have increased NADPH levels and coincided both in timing and magnitude with development of the reactive oxygen species (ROS) burst. This result indicated that plant NADK may be a downstream target which potentiates ROS production by altering NAD(H)/NADP(H) homeostasis. Further study indicated that NADK is involved in the CaM signaling pathway meditated plant oxidative burst and the hypersensitive response (Harding and Roberts, 1998).

Multiple CaM isoforms with minor amino acid sequence differences have been identified in plants. Lee et al. (1995) found two kinds of CaMs (SCaM-1 and SCaM-4) from soybean which had different effects on the Ca²⁺/CaM dependent NADK purified from pea seedlings, one is highly conserved soybean SCaM-1 which could activate the NADK, but its divergent isoform SCaM-4 could not. By generating a series of chimeric SCaMs with exchanging functional domains between SCaM-1 and SCaM-4, they found that domain I of CaM plays a key role in differential activation of NADK and the residues Lys-30 and Glu-40 of SCaM-1 are critical for this function (Lee et al., 1997). Then, Cobb et al. (1999) found that the QNP sequence in a loop between EF hands I and II in CaM is necessary for NADK activation. In tobacco, it was found that three types of CaM isoforms differentially activate NADK activity, the activity was activated most effectively by type II, moderately by type I, but weakly by type III (Karita et al., 2004). More recently, it was proved that SCaM-1 and SCaM-4 could differentially regulate NADK activity, activated by SCaM-1 but inhibited by SCaM-4 because of their different responses to the Ca²⁺ signal (Walton et al., 2017).

The activity of NADK can be regulated by altering the activity of activator CaM, but how CaM regulates NADK remains unclear. In Arabidopsis thaliana, Turner et al. (2004) found that AtNADK2 can interact with CaM, and a 45-residue region within the N-terminal domain was identified as the Ca²⁺-dependent CaM binding domain, but the recombinant AtNADK2 was not responsive to CaM in vitro. Love et al. (2015) proposed a Ca²⁺/CaM involved animal NADP synthesis model. In this model, SpNADK2 could be directly regulated by Ca²⁺/CaM binding, and SpNADK1 could be phosphorylated by CaM-dependent protein kinase II. However, further studies are needed to clarify whether a similar mechanism is functioning in plants.

Since CaMs are effective activators for the Ca²⁺/CaM-dependent NADKs, inhibition of CaMs activity is also a strategy for the inhibition of NADK activity. A series of phytotoxins from the fungus Guanomyces polytrix have been shown to inhibit NADK activity by reducing the activity of CaM (Mata et al., 2003).

NADK can catalyze NAD or NADH and ATP to synthesize NADP or NADPH, but its activity can be affected by competitive substrates which contribute to the NADP(H)/NAD(H) homeostasis in cells. In spinach, NADH was found to be a potent competitive inhibitor of NADK and the K_i (competitive inhibition constants) was 1 × 10⁻⁴ M (Yamamoto, 1966). In addition, the activity of recombinant NADHK (AtNADK3) was inhibited by poly-P_i. Mixed inhibition was observed regarding either ATP or NADH, indicating that poly-P_i may function at an allosteric site. The K_i and K_i′ (uncompetitive inhibition constants) of the NADHK for ATP and NADH are 0.23 and 1.21 mM, and 0.51 and 2.86 mM, respectively (Turner et al., 2005).

NADKs from human or microbes were also regulated by effectors like NAD(H) and NADP(H), even though the specific regulator for each NADK is different. Purified human NADK, which was expressed in E. coli, exhibited the inhibitory effects of NADPH and NADH but not of NADP (Ohashi et al., 2011). In E. coli and S. enterica, NADH and NADPH were also found to be effective inhibitors to NADK (Zerez et al., 1987; Grose et al., 2006). It was reported that the activity of NADK can be inhibited by both NADP and NADPH in Sphingomonas sp. A1 (Ochiai et al., 2004) but only strongly inhibited by NADP in Bacillus licheniformis, Bacillus subtilis and M. tuberculosis (Zerez et al., 1986; Garavaglia et al., 2003; Raffaelli et al., 2004). In M. tuberculosis and M. flavus, however, the activity can be inhibited by a low concentration of NADPH (Kawai et al., 2000). NADK activity can also be inhibited by NAD analogs in eukaryotic organisms. In S. cerevisiae, it was found that the NADK activity of Utr1p was strongly inhibited by NADP, NADH and NADPH (Kawai et al., 2001b), whereas the activity of Yef1p was only slightly inhibited by NADH and NADPH (Shi et al., 2005). Recently, Paoletti et al. (2016) found that a series of NAD mimics such as 8-thioalkyladenosine derivatives, can act as competitive substrates to inhibit Listeria monocytogenes NADK1 and Staphylococcus aureus NADK as well as their human counterpart, implying a similar allosteric regulation mode of these NADKs. In some specific situations, the activity of NADK can also be inhibited through the interaction with other enzymes. For example, ANKtk, an ATP/NAD kinase from Thermococcus kodakarensis KOD1 can bind to oxidized NOXtk hexamers which can inhibit NADK catalytic activity (Jia et al., 2010). NOXtk is a NADH oxidase from T. kodakarensis KOD1, it can be inhibited by oxidation and both unoxidized and oxidized NOXtk can form dimers and hexamers (Jia et al., 2010). However, no more evidence was provided to further clarify the allosteric regulation of NADKs, particularly in plants.

NADPH is an essential cofactor used in anabolic reactions, such as lipid and nucleic acid syntheses, which require NADPH as reducing agent. The source of NADPH biosynthesis consists of a NADK-mediated direct biosynthesis pathway and a series of dehydrogenase-mediated pathways in cells. In many studies, it was shown that after the phosphorylation of NAD by NADK, the resulting NADP could be re-reduced immediately into NADPH. In addition, NADH kinase can catalyze NADH to synthesize NADPH directly. POS5 in S. cerevisiae and AtNADK3 in Arabidopsis thaliana are representatives of NADH kinases which have been well studied (Outten and Culotta, 2003; Turner et al., 2005). These two NADKs have NADH kinase activity much higher than NAD kinase activity. NADH kinase constructs the most efficacious pathway to produce NADPH.

The other pathway for NADPH production depends on the function of a series of dehydrogenases (DHs) and transhydrogenases (THs), but all of the enzymes need NADP produced by NAD kinase. NADP-dependent dehydrogenase can reduce NADP to NADPH and NADH-dependent transhydrogenases can catalyze the transfer of proton from NADH to NADP. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6GPDH) are two enzymes that participate in the pentose phosphate pathway. G6PDH is a cytosolic enzyme which can reduce NADP to NADPH while oxidizing glucose 6-phosphate (Kletzien et al., 1994). 6GPDH also can catalyze NADP to synthesize NADPH (Rosemeyer, 1987). NADP-dependent isocitrate dehydrogenase (ICDH) is a key enzyme in the citric acid cycle (Krebs cycle) which uses NADP as a cofactor to synthesize NADPH. It is localized to the cytosol as well as to the mitochondrion and the peroxisome (Ying, 2008). In addition, other NADP dependent enzymes including NADP-dependent malic enzyme (ME), aldehyde dehydrogenase (ALDH) family Ald6p, NADP-dependent glutamate dehydrogenase (NADP-GDH), and mitochondrial transhydrogenase (mTH) and NADPH:NAD oxidoreductase (Si-specific) (pntAB), which are located in the cytosol or the mitochondria, can also contribute to NADPH production (Grabowska and Chelstowska, 2003; Sauer et al., 2004; Rydström, 2006; Singh et al., 2007). The main system of NADPH generation in plants has been summarized in Figure 4, emphasizing the fundamental role of NADKs in NADPH production.

As described above, there are three NADKs existing in Arabidopsis thaliana but only AtNADK2 is located in chloroplasts. In chloroplasts, NADP as the production of NADK accepts the electrons at the last step of the light dependent reactions in photosynthesis and its reduced form NADPH also participates in carbon fixation in light independent reactions, which indicates the importance of AtNADK2 in photosynthesis. In addition, as the major enzyme for NADPH production in chloroplasts, the functions of AtNADK2 in different metabolism pathways have been well disclosed in recent years. It was found that an AtNADK2 knockout mutant (atnadk2) showed growth inhibition and smaller rosette leaves with a pale yellow color due to the reduced chlorophyll content (Chai et al., 2005). In addition, the mutant can accumulate much more Pchlide (the precursor of chlorophyll) and Mg-Protoporphyrin IX than the wild type (Chai et al., 2005). Because the conversion of Pchlide to Chlide needs NADPH as reducing energy, due to the lack of AtNADK2 expression, the contents of NADP and NADPH were obviously reduced in the mutant and thus affected chlorophyll biosynthesis (Chai et al., 2005). Moreover, the efficiency of photosynthetic electron transport and the quantum yield of open reaction centers of Photosystem II were found to be decreased in the mutant but the non-photochemical quenching attributed to energy dissipation from the xanthophyll cycle increased (Takahashi et al., 2006), suggesting the essential role of AtNADK2 for the proper photosynthetic machinery of Photosystem II and the xanthophyll cycle. On the other hand, overexpression of AtNADK2 led to higher Calvin-Bensen cycle activity, Rubisco activity, NADP(H) concentration, nitrogen assimilation, as well as glutamine and glutamate and some other amino acids production in the transgenic plants (Takahashi et al., 2009).

As the regulator of co-enzyme homeostasis, NADK also functions in many other biosynthesis reactions. Although NADK directly contributes to NADP production in cells, it seems that the concentration of NADPH is always affected. In the atnadk2 mutant, the concentration of NADPH is lower compared to the wild type (Chai et al., 2005; Takahashi et al., 2006). Since NADPH can be utilized as reducing power in many anabolic pathways and overexpression of NADK can improve NADPH concentration efficiently, overexpressing NADK becomes a smart strategy to improve the products in anabolic pathways. For instance, overexpression of AtNADK2 in rice led to the increase in the levels of amino acids and several sugar phosphates including ribose 1,5-bisphosphate compared with the wild type (Takahara et al., 2010). The transgenic lines also showed higher electron transport and carbon dioxide (CO₂) assimilation rates (Takahara et al., 2010) and nitrogen-rich amino acids biosynthesis under elevated CO₂ (Onda et al., 2014), implying the essential roles of NADKs in plant cell metabolism.

Evidences from microbes also proved that the yields of a series of products of interest can be elevated by overexpressing NADKs. Poly(3-hydroxybutyrate) is a kind of intracellular insoluble material forming granules in bacteria, which has excellent physical and chemical properties and has a considerable prospect in new material development. It was reported that overexpressing NADK in recombinant E. coli increased the accumulation of poly(3-hydroxybutyrate) (Li et al., 2009; Zhang et al., 2015). This method was also utilized to improve the yield of a series of chemical products like L-ornithine and L-isoleucine (Shi et al., 2012; Jiang et al., 2013).

In addition, overexpression of NADH kinase can also increase the yields of carbohydrates and carotenoids by improving NADPH concentration directly. Lee et al. (2013) found that the yields of guanosine diphosphate (GDP)-L-fucose and ε-caprolactone were significantly improved by overexpressing NADH kinase POS5p of S. cerevisiae in E. coli. A similar result could be also obtained by improving carotenoid production. Glucose-6-phosphate dehydrogenase (Zwf1) and the NADH kinase Pos5 are the two main NADPH-supplying sources in S. Cerevisiae, overexpression of Pos5 led to 85 and 39% of lycopene and β-carotene increases in the recombinant S. cerevisiae, respectively, as compared to the Zwf1 overexpressed strain. Overexpressing Pos5 is more effective than overexpressing Zwf1 for the production of carotenoids (Zhao et al., 2015).

For further improving the yield of products of interest, a more comprehensive and valid method has been adopted. Since both NADK and NADP-dependent dehydrogenases can improve the concentration of NADPH, overexpression of the two kinds of enzymes simultaneously should be expected to results in elevated NADPH production. In an engineered shikimic acid-producing E. coli strain, it was reported that the increasing NADPH availability, achieved by overexpressing pntAB (a transhydrogenase gene) and/or NADK genes, significantly enhanced shikimic acid production (Cui et al., 2014). Isobutanol is an alternative biofuel. Shi et al. (2013) found that overexpressing both transhydrogenase (pntAB) and NAD kinase (yfjB) genes significantly increased isobutanol production in recombinant E. coli. Using the same strategy can also promote the production of free fatty acids (Wu et al., 2014).

Improving the level of the reducing power is an efficient strategy to resist the oxidative stresses provoked by environmental stimulation, which results in NADK having an indispensable role in responding to environmental stresses. NADK activity of plants has variations under different environmental stresses. In wheat, NADK activity was decreased in leaves by water-deficit stress (Zagdanska, 1990) but increased in apical segments of roots after exposure to aluminum (Slaski, 1995). When treated by indoleacetic acid (IAA), its activity in wheat coleoptile was up-regulated (Li et al., 1999). Under conditions of cold-shock stress, the Ca²⁺-dependent NADK activity in green bean leaves was raised (Ruiz et al., 2002). In strawberry, Gu (2007) found that ethylene can down-regulate the activity of NADK, reducing the concentration of NADPH, which accelerates the ripening of fruit indirectly. But under cold environmental conditions, the activity was raised, leading to an increase in NADPH concentration to prevent the fruits from ripening (Gu et al., 2007).

In addition, many studies have shown that the expression of NADK family genes can be strongly stimulated by a number of environmental stresses in plants. The expression of A. thaliana AtNADK1 was up-regulated 3–8-fold under several treatments including ionizing radiation, hydrogen peroxide (H₂O₂) application and plant pathogen interactions (Berrin et al., 2005). AtNADK3 was also inducible by environmental treatments, its expression was up-regulated more than 5-fold by methyl viologen (MV) treatment and raised 3-fold by NaCl treatment (Chai et al., 2006). When treated with mannitol or low temperature, it had 6-fold and 8-fold up-regulation, respectively (Chai et al., 2006). Although there are some species specificities between the orthologous and/or paralogous genes of the cereal NADK genes, their transcripts in wheat, rice and maize were all strongly affected by one or several environmental stress treatments such as cold, heat, dehydration (PEG), salt (NaCl), oxidative stress (MV), and hormones including abscisic acid (ABA), methyl jasmonic acid (MeJA), and salicylic acid (SA) (Wang et al., 2016), implying their crucial roles in the abiotic stress response and hormone signaling regulation.

To better understand the functions of NADK family genes in plant stress responses, the inducible expression patterns of NADK family genes from eight kinds of plants have been summarized here based on the microarray and mRNA sequencing data from Genevestigator (Table 3, Figure S3). The results show that the expression of NADK family genes was not only induced by abiotic stresses and hormone treatments, but also induced by biotic stresses. For instance, TaNADK1s, TaNADK3s, and TaNADK4s were down-regulated but TaNADK2s were up-regulated under F. graminearum attack. TaNADK2s were also up-regulated under P. graminis attack. According to the expression profiles of eight kinds of NADK family genes including AtNADKs, OsNADKs, TaNADKs, ZmNADKs, HvNADKs, MtNADKs, GmNADKs, and SlNADKs, NADK family genes from different species were induced to varying degrees by abiotic/biotic stresses and hormone treatments, exhibiting high diversity in response to various environmental stresses between the orthologous and/or paralogous genes (Table 3, Figure S3). Besides, the expression level of a particular NADK has a different response to different concentrations of a certain treatment. For example, AtNADK3 transcripts were up-regulated more than 5-fold after a 6 h treatment with 1 μM MV, but they were down-regulated deeply after a 12 h treatment with 30 μM MV, implying its spatiotemporal expression character. Although we could not exactly distinguish the certain roles of each NADK gene in plant stress tolerance at the current stage, all of these results underscore the functional diversity and divergence of NADK family genes of plants in responding to a number of abiotic and biotic stresses.

During plant growth, ROS are produced as metabolic by-products. However, when plants experience different kinds of environmental stresses including abiotic stresses like cold, heat, salt, drought, and also biotic stresses including different pathogen attacks, ROS are produced as signaling components in stress tolerance (Neill et al., 2002; Edreva, 2005; Foyer and Noctor, 2005; Noctor and Foyer, 2016). Although low levels of ROS play an important role in signaling events, excessive ROS are harmful for plants. They can lead to cell death by oxidative processes including membrane lipid peroxidation, protein oxidation, enzyme inhibition, and DNA and RNA damage (Mittler, 2002; Circu and Aw, 2010; Liu and He, 2016). The excess ROS induced by environmental stresses must be scavenged. Therefore, ROS homeostasis is very important for the survival of plants under adverse environmental conditions.

To demonstrate the importance of NADK in ROS scavenging systems, we summarized here the main sources of ROS producing systems, anti-oxidative systems and NADPH supplying systems in different subcellular compartments of plants (Figure 2). In the cytosol, the plasma membrane-targeted NADPH oxidases (NOXs) can produce ROS by utilizing intracellular NADPH as reductant (Bedard et al., 2007). Chloroplasts and mitochondria are not only powerhouses, but also the main ROS producers in plant cells because of photosynthetic and respiratory electron transfer chains, respectively (Asada and Takahashi, 1987; Asada, 1999; Maxwell et al., 1999; Dat et al., 2000). Excited chlorophyll can also generate singlet oxygen (¹O₂), a high-energy form of oxygen (Asada and Takahashi, 1987). In peroxisomes, ROS can be generated from several ways including glycolate oxidation, fatty acid β-oxidation, and xanthine oxidation (Corpas et al., 2001). On the other hand, the ascorbate-glutathione cycle (Halliwell-Asada cycle) and related enzymes including glutathione reductase (GR), dehydroascorbate reductase (DHAR), glutathione dehydrogenase (GDH) and ascorbate peroxidases (APX) play crucial roles in the scavenge of ROS induced by environmental stresses (Noctor and Foyer, 1998; Horemans et al., 2000; Foyer et al., 2006). Besides, carotenoids and catalase also can scavenge ROS efficiently in chloroplasts and peroxisomes, respectively (Asada and Takahashi, 1987; Willekens et al., 1997). As the only known enzyme to synthesize NADP(H), NADK is indispensable in anti-oxidative stress systems because NADP(H) can provide the main reduction power for the ROS scavenging system (Mittler, 2002) and therefore NADK plays crucial roles in ROS homeostasis of plant cells. Ca²⁺/CaM also played a dual role in NOX-mediated ROS production and NADPH dependent scavenging (Figure 2). The activation of NOX needs Ca²⁺ binding to its EF-hand motif and the phosphorylation by Ca²⁺ dependent protein kinase, such as calcium-dependent protein kinase (CDPK), calmodulin (CaM), and calmodulin-like protein (CML) or calcineurin B-like protein (CBL) (Kobayashi et al., 2007; Ogasawara et al., 2008; Drerup et al., 2013; Dubiella et al., 2013). Roberts and Harmon (1992) reviewed how CaMs contribute to Ca²⁺/CaM dependent NADK activation in higher plants. This further emphasizes the significance of NADK in ROS homeostasis.

As described above, NADK isoforms have multiple subcellular localizations, which can supply sufficient NADPH in different subcellular compartments in plants. In chloroplasts, NADPH not only participates in NADPH-dependent anti-oxidative systems directly, but also tightly relates to carotenoids biosynthesis (Hornero-Méndez and Britton, 2002). As a chloroplast-targeted NADK in Arabidopsis thaliana, AtNADK2 is essential in maintaining the NADPH levels of chloroplasts. Takahashi et al. (2006) found that the atnadk2 mutant showed an aberrant de-epoxidation state of its xanthophyll cycle carotenoids which may relate to the pale green phenotype and growth inhibition of the mutant plants. In cyanobacteria, MV treatment can damage macromolecules and membranes by accepting electron from Photosystem I and reducing O₂ to O2-. It was reported that the sll1415 mutant (a kind of NADK knockout mutant) exhibited enhanced sensitivity to MV treatments, indicating that the NADK is essential to resist oxidative stress (Gao and Xu, 2012). In peroxisomes, NADK (like AtNADK3 and TaNADK4) was suggested not only to participate in the balancing of NAD(H) and NADP(H) but also to function in fatty acid β-oxidation (Waller et al., 2010; Wang et al., 2016). All these results suggest that NADK plays a crucial role in ROS homeostasis in different subcellular compartments of plants. By improving the NADPH level efficiently, NADK functions in both ROS generation and scavenging through NADPH-dependent NOXs and NADPH-dependent scavenging systems, respectively.

NADPH/NADP, AsA (ascorbate)/DHA (dehydroascorbate) and GSH (glutathione)/GSSG (oxidized glutathione) are the three major intracellular redox couples which are widely involved in metabolism, signal transduction and transcriptional regulation of plants (May et al., 1998; Pollak et al., 2007a; Bilan et al., 2015). Due to the vital roles of NADK in the conversion of NAD to NADP and the synthesis of NADPH, it firstly contributes to the balance of NAD(H) and NADP(H) in cells. It was reported that the metabolic redox balance between NADH and NADPH is essential for cell survival under oxidative stress and the increase in NADPH by NADK is needed by cells to protect against oxidative challenges in Pseudomonas fluorescens (Mailloux et al., 2011). Gray et al. (2012) found that NADK regulates the ratio of NADPH/NADP and affects the intracellular redox and oxidative defense in the pancreatic β-cells. In Arabidopsis thaliana, Berrin et al. (2005) found that AtNADK1 may contribute to the maintenance of redox status in cells to withstand gamma irradiation and MV-induced oxidative stress. AtNADK2 also functions in the balance of NAD(H) and NADP(H) to cope with environmental stresses in the plant. It was found that, the content of NAD(H) was increased but that of NADP(H) was decreased in the AtNADK2 deletion mutant atnadk2: consequently, the tolerance of the mutant to various environmental stresses such as UVB, drought, heat and salt was markedly reduced (Chai et al., 2005; Takahashi et al., 2006, 2009; Takahara et al., 2010). In addition, previous studies showed that A. thaliana AtNADK3 is a peroxisomal enzyme (Waller et al., 2010) and preferentially phosphorylates NADH over NAD to yield NADPH in the plant (Turner et al., 2004, 2005). More recently, we found that lack of OsNADK1, a cytosolic NADK in rice, significantly increased the ratio of NAD(H)/NADP(H) but decreased the ratio of NADPH/NADP, implying an important role of OsNADK1 in the balance of NAD(H) and NADP(H) of rice (data not shown).

As the two other intracellular redox couples, roles of AsA/DHA and GSH/GSSG in plant stress responses or tolerance were paid great attention in the past decades (May et al., 1998). Li and van Staden (1998) found that the content of AsA was decreased in maize under drought treatment. However, Schwanz et al. (1996) reported that the total AsA was decreased but the reduction AsA was increased in oak trees under drought conditions. In the roots of tomato, Shalata et al. (2001) found that the content of AsA was decreased but DHA increased, leading to the ratio of AsA/DHA in a salt sensitive cultivar being decreased under salt stress conditions. In other reports, it was shown that GSH/GSSG ratios might affect the whole redox potential of cells by influencing NAD/NADP metabolism. Decreased GSH/GSSG ratios lead to decreased NADPH/NADP ratios, and the decreased NADPH/NADP ratios may affect NADH/NAD ratios due to the NADKs modulating capacity (Ying, 2008). In fact, a knockout mutant of A. thaliana AtNADK3, atnadk3, showed a decrease of NADPH, and also exhibited a decreased ratio of GSH/GSSG (Chai et al., 2006). In our study, a marked decrease in the ratios of AsA/DHA and GSH/GSSG was also observed in a rice OsNADK1-knockout mutant (data not shown). All these results strongly suggest that NADK plays crucial roles in the maintaining intracellular redox balance of plants.

Several reports have illustrated that NADK deficient mutants become more sensitive to environmental stresses in Arabidopsis thaliana. When treated by MV a potent oxidant, AtNADK1-deficient mutant plants can produce a high level of anthocyanin, a regulator when plants are subjected to oxidative stress, indicating that the atnadk1 mutant is more sensitive to oxidative stress (Berrin et al., 2005). In an AtNADK2-deficient mutant, the total NADPH level is lower than wild type, which also displayed hypersensitivity to oxidative stress induced by UVB, drought, heat shock or salinity (Chai et al., 2005). The AtNADK3-deficient mutant also showed hypersensitivity to oxidative stress in both seed germination and seedling growth (Chai et al., 2006). In contrast, overexpressing NADK confers tolerance to oxidative stress. In rice overexpressing AtNADK2 transgenic lines, the ratio of GSH/GSSG was significantly elevated, which led to a slower decrease in the photochemical efficiency of Photosystem II and total chlorophyll, and a lower concentration of malondialdehyde, a marker of lipid peroxidation, indicating a higher stress tolerance of the transgenic plants (Takahara et al., 2010). Petriacq et al. (2012) also proved that higher NADPH levels can enhance the tolerance of Arabidopsis thaliana transgenic plants to the pathogen Pseudomonas syringae pv. tomato Pst-AvrRpm1. Recently, we found that overexpression of OsNADK1 in rice led to high tolerance of the plant to drought stress. The transgenic plants exhibited a high content of proline and transcripts of several redox-sensitive genes such as OsDREB1B, whereas the loss of function plants of OsNADK1 are opposite in this regard (data not shown). Overexpression of wheat TaNADK4 in rice also confers to the plant a strong tolerance to drought (data not shown).

Interestingly, due to the essential roles in ROS homeostasis and cell survival under stress conditions, NADK has been considered as a drug target in anti-cancer medicine research. Recently, two different strategies were used to restrain the growth of cancer cells by inhibiting NADK activity. Dihydrofolate reductase is an essential enzyme involved in de novo purine and thymidine biosynthesis but needs NADPH for the activity. Benzamide riboside, a drug targeted to NADK, can reduce NADP and NADPH levels, and therefore treatment with this drug can lead to the process of DNA synthesis and growth of cancer cells being inhibited (Roussel et al., 2012). Thionicotinamide adenine dinucleotide phosphate is also used as an inhibitor of NADK to accelerate the degradation of dihydrofolate reductase and thus to inhibit the growth of cancer cells (Hsieh et al., 2013). The other strategy is to inhibit proliferation by inducing high level of ROS in cancer cells. NADPH is essential for the neutralization of the dangerously high levels of ROS generated by increased metabolic activity of cancer cells. Inhibition of NADK activity with shRNA or thionicotinamide increased the ROS production in cancer cells which led to synergistic cell death (Tedeschi et al., 2015, 2016). These results demonstrate the value of NADK in anti-cancer research, and at the same time give us an attractive cue to use similar approaches for stress tolerance of plants.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.