ORIGINAL ARTICLE Subcellular and tissue localization of NAD kinases from Arabidopsis: compartmentalization of de novo NADP biosynthesis Jeffrey C. Waller • Preetinder K. Dhanoa • Uwe Schumann • Robert T. Mullen • Wayne A. Snedden Received: 10 August 2009 / Accepted: 21 October 2009 / Published online: 17 November 2009 Ó Springer-Verlag 2009 Abstract The de novo biosynthesis of the triphospho- pyridine NADP is catalyzed solely by the ubiquitous NAD kinase family. The Arabidopsis (Arabidopsis thaliana) genome contains two genes encoding NAD ? kinases (NADKs), annotated as NADK1, NADK2, and one gene encoding a NADH kinase, NADK3, the latter isoform preferring NADH as a substrate. Here, we examined the tissue-specific and developmental expression patterns of the three NADKs using transgenic plants stably trans- formed with NADK promoter::glucuronidase (GUS) reporter gene constructs. We observed distinct spatial and temporal patterns of GUS activity among the NADK::GUS plants. All three NADK::GUS transgenes were expressed in reproductive tissue, whereas NADK1::GUS activity was found mainly in the roots, NADK2::GUS in leaves, and NADK3::GUS was restricted primarily to leaf vasculature and lateral root primordia. We also examined the subcel- lular distribution of the three NADK isoforms using NADK–green fluorescent protein (GFP) fusion proteins expressed transiently in Arabidopsis suspension-cultured cells. NADK1 and NADK2 were found to be localized to the cytosol and plastid stroma, respectively, consistent with previous work, whereas NADK3 localized to the peroxi- somal matrix via a novel type 1 peroxisomal targeting signal. The specific subcellular and tissue distribution profiles among the three NADK isoforms and their possible non-overlapping roles in NADP(H) biosynthesis in plant cells are discussed. Keywords NADH kinase Á NAD kinase Á Peroxisome Á Plant Á Pyridine nucleotide Á Subcellular localization Introduction Since their discovery over 100 years ago, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) have become well estab- lished as key energy transducers, precursors for the sig- naling molecules cADPR and NAADP, and metabolic regulators (reviewed in Noctor et al. 2006). For example, there are *800 oxidoreductases predicted in the Arabid- opsis thaliana (Arabidopsis) genome which likely use the pyridine nucleotide cofactors NAD or NADP (Kho et al. 2003). In addition, these cofactors are well known to affect In this article, NAD(P) is used to denote NAD or NADP without regard to its reduction status or in cases where the distinction between the two forms is unnecessary (e.g. NADP/NAD ratios). NAD(P) ? and NAD(P)H are used to make specific reference to the oxidized forms and reduced forms, respectively. Electronic supplementary material The online version of this article (doi:10.1007/s00425-009-1047-7) contains supplementary material, which is available to authorized users. J. C. Waller Á W. A. Snedden (&) Department of Biology, Queen’s University, Kingston, ON K7L 3N6, Canada e-mail: sneddenw@queensu.ca P. K. Dhanoa Á U. Schumann Á R. T. Mullen Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada Present Address: J. C. Waller Horticultural Sciences Department, University of Florida, Gainesville, FL 32611-0690, USA Present Address: U. Schumann Institut fu ¨r Molekulare Herz-Kreislaufforschung (IMCAR), Universista ¨tklinikum Aachen (RWTH), Pauwelsstrasse 30, 52074 Aachen, Germany 123 Planta (2010) 231:305–317 DOI 10.1007/s00425-009-1047-7