NADK2, an Arabidopsis chloroplastic NAD kinase, plays a vital role in both chlorophyll synthesis and chloroplast protection Mao-Feng Chai, Qi-Jun Chen, Rui An, Ye-Miao Chen, Jia Chen and Xue-Chen Wang* National Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricul- tural University, 100094, Beijing, China (*author for correspondence; e-mail xcwang@cau.edu.cn) Received 27 October 2004; accepted in revised form 29 April 2005 Key words: chlorophyll synthesis, chloroplast, chloroplast protection, NAD kinase Abstract As one of terminal electron acceptors in photosynthetic electron transport chain, NADP receives electron and H + to synthesize NADPH, an important reducing energy in chlorophyll synthesis and Calvin cycle. NAD kinase (NADK), the catalyzing enzyme for the de novo synthesis of NADP from substrates NAD and ATP, may play an important role in the synthesis of NADPH. NADK activity has been observed in different sub-cellular fractions of mitochondria, chloroplast, and cytoplasm. Recently, two distinct NADK isoforms (NADK1 and NADK2) have been identified in Arabidopsis. However, the physiological roles of NADKs remain unclear. In present study, we investigated the physiological role of Arabidiposis NADK2. Sub-cellular localization of the NADK2–GFP fusion protein indicated that the NADK2 protein was localized in the chloroplast. The NADK2 knock out mutant (nadk2) showed obvious growth inhibition and smaller rosette leaves with a pale yellow color. Parallel to the reduced chlorophyll content, the expression levels of two POR genes, encoding key enzymes in chlorophyll synthesis, were down regulated in the nadk2 plants. The nadk2 plants also displayed hypersensitivity to environmental stresses provoking oxidative stress, such as UVB, drought, heat shock and salinity. These results suggest that NADK2 may be a chloroplast NAD kinase and play a vital role in chlorophyll synthesis and chloroplast protection against oxidative damage. Introduction NADP is essential for biosynthetic pathways, energy metabolism, and signal transduction in living organisms (Garavaglia et al., 2003). NAD kinase (NADK, EC 2.7.1.23) catalyzes the NADP formation from substrates NAD and ATP through phosphorylation. This enzyme has been regarded as the sole catalyzer for the de novo biosynthesis of NADP (Kawai et al., 2001a), suggesting a regula- tory role in the NADP-dependent anabolic/bio- synthetic pathways. NADKs have been found in all organisms investigated to date, suggesting an essential role in all organisms (McGuinness and Butler, 1985; Zielinski, 1998). They may be the key enzymes in the synthesis of NADPH by providing NADP. NADPH plays important roles in energy metabolism and reductive biosynthesis in all organisms. In chloroplast, NADPH, produced from NADP in photosynthetic electron transport chain, provides important reducing energy in chlorophyll (chl) synthesis and Calvin cycle. In plants, NADK has been found in chlorop- lasts (Muto et al., 1981; Jarrett et al., 1982), mitochondria (Dieter and Marme, 1984) and cytoplasm (Simon et al., 1982). Moreover, plants possess both calmodulin (CaM)-independent and CaM-dependent NADK isoforms (Roberts and Plant Molecular Biology (2005) 59:553–564 Ó Springer 2005 DOI 10.1007/s11103-005-6802-y