| Abstract: | The chloroplastic NAD kinase (NADK2) is reported to stimulate carbon and nitrogen assimilation in Arabidopsis (Arabidopsis thaliana), which is vulnerable to high light. Since rice (Oryza sativa) is a monocotyledonous plant that can adapt to high light, we studied the effects of NADK2 expression in rice by developing transgenic rice plants that constitutively expressed the Arabidopsis chloroplastic NADK gene (NK2 lines). NK2 lines showed enhanced activity of NADK and accumulation of the NADP(H) pool, while intermediates of NAD derivatives were unchanged. Comprehensive analysis of the primary metabolites in leaves using capillary electrophoresis mass spectrometry revealed elevated levels of amino acids and several sugar phosphates including ribose-1,5-bisphosphate, but no significant change in the levels of the other metabolites. Studies of chlorophyll fluorescence and gas change analyses demonstrated greater electron transport and CO2 assimilation rates in NK2 lines, compared to those in the control. Analysis of oxidative stress response indicated enhanced tolerance to oxidative stress in these transformants. The results suggest that NADP content plays a critical role in determining the photosynthetic electron transport rate in rice and that its enhancement leads to stimulation of photosynthesis metabolism and tolerance of oxidative damages.NADP is a ubiquitous coenzyme, required in various metabolic processes, since these metabolites carry electrons through the reversible conversion between oxidized (NAD+, NADP+) and reduced (NADH, NADPH) forms in all organisms. NAD is highly oxidized and is involved primarily in intracellular catabolic reactions, whereas NADP is predominantly found in its reduced form and participates in anabolic reactions and defense against oxidative stress (Ziegler, 2000; Noctor et al., 2006; Pollak et al., 2007a). Since NAD(H) and NADP(H) play a variety of distinct physiological roles, the regulation of the NAD(H)/NADP(H) balance is essential for cell survival (Kawai and Murata, 2008; Hashida et al., 2009).One of the key enzymes that regulates NAD(H)/NADP(H) balance is NAD kinase (NADK; EC 2.7.1.23), which catalyzes NAD phosphorylation in the presence of ATP. The genes encoding NADK were cloned recently from all organisms investigated to date, except for Chlamydia trachomatis (Kawai and Murata, 2008). Only a single gene encoding NADK has been found in some bacteria and mammals (Kawai and Murata, 2008). In contrast, NADK activity was detected in not only the cytosol but also organelles in yeast and plant (Jarrett et al., 1982; Simon et al., 1982; Dieter and Marme, 1984; Iwahashi and Nakamura, 1989; Iwahashi et al., 1989), and three genes including cytosol-type and organelle-type NADK have been cloned in yeast (Kawai et al., 2001; Outten and Culotta, 2003) and plants (Turner et al., 2004, 2005).In Arabidopsis (Arabidopsis thaliana), one of the NADK isoforms is localized in the chloroplast (NADK2; Chai et al., 2005), the others are localized in the cytosol (NADK1 and NADK3; Chai et al., 2006). Analysis of Arabidopsis mutants revealed low chlorophyll (chl) content, low photosynthetic activity, growth inhibition, and hypersensitivity to environmental stresses in the nadk2 knockout mutant (Chai et al., 2005; Takahashi et al., 2006), whereas the nadk1 knockout mutant and the nadk3 knockout mutant did not show a significant phenotype, except for sensitivity to oxidative stress (Berrin et al., 2005; Chai et al., 2006). Moreover, the major part of NADP(H) biosynthesis in photosynthetic organ appears to be attributable to NADK2, because NADK and NADP(H) were strictly decreased in leaves of the nadk2 knockout mutant (Chai et al., 2005; Takahashi et al., 2006). In the plant cell, NADP is mainly located in the chloroplast (Heber and Santarius, 1965; Wigge et al., 1993), where NADP+ functions as the final electron acceptor of the photosynthetic electron transport. The reducing energy obtained is not only supplied for Calvin cycle, nitrogen assimilation, lipid and chl metabolism, but also play a crucial role in maintaining redox homeostasis through the regulation of producing and consuming reactive oxygen species (ROS) in the plant cell (Noctor, 2006; Noctor et al., 2006). Accordingly, these evidences indicate that chloroplastic NADK2 plays a central role in plant metabolism and stress tolerance through homeostasis of ROS as regulator of NADP/NAD balance.Since the alteration of NAD/NADP balance affects metabolism and ROS homeostasis, manipulation of NADK can be an attractive target for the engineering of plant metabolism. It was reported that overexpression of NADK causes perturbation of NADP(H) pool and has positive effects on stress tolerance or growth in various living things. In Asperadium nidulans, overexpression of NADH kinase improves the growth efficiency of the cell (Panagiotou et al., 2009). Overexpression of NADK in human HEK293 cells causes 4-to 5-fold increase of NADPH concentration and provides moderate protection against oxidative stress (Pollak et al., 2007b). Recently, we evaluated effects of the enhanced NADP(H) content in Arabidopsis by generating NADK2-overexpressing plants (Takahashi et al., 2009). Our results indicated that enhanced NADP(H) production by NADK2 overexpression promoted nitrogen assimilation and resulted in accumulation of metabolites associated with the Calvin cycle, accompanied by increased activity of Rubisco. Together, these studies demonstrated the potential use of NADK as candidate gene in promoting primary metabolism and/or stress tolerance in transgenic plants.Rice (Oryza sativa) is not only the primary crop for more than half of the world''s population, but also a model monocot system. Rice can adapt to more strong light intensity than Arabidopsis, because rice is a sun plant, whereas Arabidopsis is a shade plant. Therefore, it is possible that effects of an increased NADP(H) content could be more significant in rice plant than in Arabidopsis, due to a higher ability to manage reductive energy involved in NADP as an electron carrier. In this article, we describe the generation and characterization of transgenic rice plants expressing an Arabidopsis chloroplastic NADK (AtNADK2), under the control of the maize (Zea mays) ubiquitin promoter. We named the rice plant as NK2. We found that pleiotropic effects on primary metabolism in NK2 rice were similar to the result obtained in NADK2-overexpressing Arabidopsis plants. However, stimulation of carbon fixation and nitrogen assimilation were observed in NK2 rice, accompanying with significant increases in electron transport and CO2 assimilation rates, unlike results of the previous study of Arabidopsis. Interestingly, the NK2 lines also showed enhanced tolerance to oxidative stress. |
