Subcellular and tissue localization of NAD kinases from Arabidopsis: compartmentalization of de novo NADP biosynthesis

Abstract

The de novo biosynthesis of the triphosphopyridine 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 transformed 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 subcellular 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 peroxisomal 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.

Defects in Peroxisomal 6-Phosphogluconate Dehydrogenase Isoform PGD2 Prevent Gametophytic Interaction in Arabidopsis thaliana

“…Hence, in the amc mutant, NADPH formation inside peroxisomes should be abolished. This seems to primarily affect the OPPP branch (comprising G6PD1, PGL3, and PGD2; Meyer et al, 2011) as well as other enzymes with a PTS1 motif, like NADP-ICDH (Leterrier et al, 2012) or NADK3 (Waller et al, 2010).…”

“…Recently, NADPH production in peroxisomes was shown to be important for the biosynthesis of nitric oxide (NO) in roots facing abiotic stress, either induced by salt (NaCl; Corpas et al, 2009) or heavy metals (cadmium; Corpas and Barroso, 2014a). Whether peroxisomal NADPH may be needed for NO synthesis, which plays an important role during fertilization (Prado et al, 2004(Prado et al, , 2008, remained unclear, mainly because, aside from the OPPP, NADPH in peroxisomes also can be produced by other enzymes, such as NADP-dependent isocitrate dehydrogenase (NADP-ICDH; At1g54340) with peroxisomal targeting signal type 1 (PTS1) motif SRL Leterrier et al, 2012) or stress-related phosphorylation of NAD(H) via the NAD kinase isoform NADK3 (Chai et al, 2006) with PTS1 motif SRY (Waller et al, 2010). Hence, under which conditions OPPP reactions in peroxisomes may be necessary or even essential remained to be determined.…”

Identification of Novel Plant Peroxisomal Targeting Signals by a Combination of Machine Learning Methods and in Vivo Subcellular Targeting Analyses

“…The novel PTS1 tripeptide, SRY> 1 , had been identified independently by Waller et al (2010). Three additional decapeptides investigated in this study represented putative (and validated) non-PTS1 sequences (LCR>, LNL>, and APN>) and are not listed (see Supplemental Tables 1 and 5 online).…”

“…Moreover, the era of experimental research on low-abundance peroxisomal matrix proteins and characterization of their atypical PTS1 tripeptides has begun only recently. EST database searches for putatively orthologous plant sequences using the Arabidopsis proteins identified in this study (see Supplemental Table 5 online) and others with noncanonical PTS1s, such as Arabidopsis glutathione reductase (TNL>; Kataya and Reumann, 2010) and NADK3 (SRY>; Waller et al, 2010), will certainly allow the recognition of further noncanonical PTS1 tripeptides.…”

The Potato Tuber Mitochondrial Proteome

“…We here identify a NAD + carrier as well as a NADH kinase (Supplemental Table S1). Three NAD(H) kinases are found in Arabidopsis, and one of them, nucleoside diphosphate kinase3, which appears to prefer NADH as substrate over NAD + , has been reported to be localized to peroxisomes in higher plants, while its analog in yeast is mitochondrial (Waller et al, 2010). The POM NADH kinase is predicted to be mitochondrial by three prediction programs and is of relatively low abundance (23.87 [log 10 (dNSAF)]; Fig.…”