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First published online August 19, 2005; 10.1104/pp.105.065953 Plant Physiology 139:39-51 (2005) © 2005 American Society of Plant Biologists A Comprehensive Analysis of the NADP-Malic Enzyme Gene Family of Arabidopsis1,[w]Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Rosario, Argentina (M.C.G.W., M.A.T., M.F.D., C.S.A.); and Botanisches Institut, Universität zu Köln, D–50931 Cologne, Germany (U.-I.F., V.G.M.)
The Arabidopsis (Arabidopsis thaliana) genome contains four genes encoding putative NADP-malic enzymes (MEs; AtNADP-ME1–ME4). NADP-ME4 is localized to plastids, whereas the other three isoforms do not possess any predicted organellar targeting sequence and are therefore expected to be cytosolic. The plant NADP-MEs can be classified into four groups: groups I and II comprising cytosolic and plastidic isoforms from dicots, respectively; group III containing isoforms from monocots; and group IV composed of both monocots and dicots, including AtNADP-ME1. AtNADP-MEs contained all conserved motifs common to plant NADP-MEs and the recombinant isozymes showed different kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and NADP-ME4 present the highest catalytic efficiency for NADP and malate, respectively. NADP-ME4 exists in equilibrium of active dimers and tetramers, while the cytosolic counterparts are present as hexamers or octamers. Characterization of T-DNA insertion mutant and promoter activity studies indicates that NADP-ME2 is responsible for the major part of NADP-ME activity in mature tissues of Arabidopsis. Whereas NADP-ME2 and -ME4 are constitutively expressed, the expression of NADP-ME1 and NADP-ME3 is restricted by both developmental and cell-specific signals. These isoforms may play specific roles at particular developmental stages of the plant rather than being involved in primary metabolism.
Malic enzymes (MEs) catalyze the oxidative decarboxylation of L-malate, producing pyruvate, CO2, and NAD(P)H in the presence of a divalent cation (Chang and Tong, 2003  ). This enzyme is widely distributed in nature due to the participation of the reaction products in a large number of metabolic pathways (Lance and Rustin, 1984). In plants, NAD-dependent isoforms (NAD-ME; EC 1.1.1.39) function predominantly in mitochondria to produce pyruvate for oxidation in the tricarboxylic acid cycle (Artus and Edwards, 1985), while NADP-dependent MEs (NADP-ME; EC 1.1.1.40) are found in cytosol and plastids (Edwards and Andreo, 1992; Drincovich et al., 2001). Thus, different NADP-ME isoforms have been found in bundle sheath chloroplasts of some C4 plants and in the cytosol of some Crassulacean acid metabolism (CAM) plants, where they take part in the photosynthetic metabolism, as well as in the cytosol and plastids of photosynthetic and nonphotosynthetic tissues of C3, C4, and CAM plants playing different postulated nonphotosynthetic roles (Drincovich et al., 2001; Lai et al., 2002a, 2002b).
The biological role of NADP-MEs, apart from being involved in C4 and CAM photosynthesis, remains elusive. Plastidic nonphotosynthetic isoforms were suggested to be involved in plant defense responses (Casati et al., 1999
The complete Arabidopsis (Arabidopsis thaliana) genome presents the opportunity to study the whole set of NAD(P)-ME isoforms present in this C3 dicot plant (Arabidopsis Genome Initiative, 2000
Cloning and In Silico Analysis of the AtNADP-ME Family
Four putative AtNADP-ME genes were identified as orthologs to the nonphotosynthetic maize NADP-ME (AY315822; Maurino et al., 2001
Within the AtNADP-ME family, the predicted amino acid sequences showed identities between 78% (NADP-ME1 versus NADP-ME4) and 91% (NADP-ME2 versus NADP-ME3). A multiple alignment with known NADP-MEs showed that all NADP-MEs, including the AtNADP-MEs, contained conserved motifs (data not shown; Drincovich et al., 2001
NADP-ME4 Is Localized to Plastids To obtain experimental evidence for the functionality of the predicted pTP of NADP-ME4, the region encoding the putative pTP was fused in frame to the green fluorescent protein (GFP) coding sequence. As shown in Figure 2A, the first 252 nucleotides of NADP-ME4 were able to direct GFP to the plastids in transient transfection assays using tobacco (Nicotiana tabacum) BY-2 protoplasts (Fig. 2A). A control using the GFP coding region shows localization of the free GFP to cytosolic and nuclear compartments (Fig. 2A).
Heterologous Expression and Biochemical Characterization of Recombinant AtNADP-ME Isoforms
To assess whether the four predicted AtNADP-ME cDNAs encode enzymatically active NADP-ME proteins, the different NADP-ME isoforms were expressed in Escherichia coli (in the case of NADP-ME4, without the pTP) and purified to homogeneity. Following induction by isopropylthio-
Figure 3B presents results obtained by native electrophoresis of the purified recombinant AtNADP-MEs. As a control, the tetrameric maize recombinant photosynthetic NADP-ME was analyzed (Detarsio et al., 2003 ). Activity staining assays indicate that NADP-ME4, the plastidic isoform, exists in equilibrium of active tetramers and dimers, as the molecular masses of the active bands are consistent with these oligomeric states (approximately 260 and 130 kD). Interestingly, the native molecular masses of the cytosolic isoforms (NADP-ME1, NADP-ME2, and NADP-ME3) are higher than the 400 kD indicative for higher oligomeric states of these isoenzymes, probably hexamers or octamers.
Recombinant purified NADP-ME1, NADP-ME2, NADP-ME3, and NADP-ME4 were characterized with respect to kinetic properties (Table I). The isoforms that presented the highest specific activities were the cytosolic forms NADP-ME2 followed by NADP-ME3. The specific activity of NADP-ME2 was more than twice as high as that obtained for NADP-ME4 and more than 8 times the value for NADP-ME1. Comparing the Km (NADP) values, NADP-ME3 and NADP-ME4 exhibit the highest affinity toward NADP (6–10 µM), followed by NADP-ME2 (approximately 70 µM), and NADP-ME1 with the lowest affinity of all NADP-MEs characterized up to now (approximately 200 µM; Drincovich et al., 2001
Expression Analysis of the Arabidopsis NADP-ME Genes
Semiquantitative reverse transcription (RT)-PCR was conducted to analyze the expression of NADP-ME in different tissues of Arabidopsis. NADP-ME2 and NADP-ME4 transcripts are present in all organs tested, whereas NADP-ME1 is preferentially expressed in roots (Fig. 2B). By contrast, NADP-ME3 transcripts are hardly detectable in roots, while mRNA abundance is higher in flowers (Fig. 2B). To further investigate the expression of AtNADP-MEs, publicly available microarray data were also analyzed (http://www.genevestigator.ethz.ch; Zimmermann et al., 2004 To allow a more thorough analysis of AtNADP-ME gene expression during plant development, translational fusions of the promoter sequences, the first exon and intron, and part of the second exon with the uidA (GUS) gene were stably introduced into Arabidopsis. All the T1 plants selected showed the same qualitative expression pattern. T2 plants from at least six independent transformants per construct were analyzed histochemically for GUS activity.
Expression during Embryogenesis
Expression during Germination The expression pattern of the four AtNADP-ME genes was analyzed during the first 2 weeks after imbibition. It is worth mentioning that, due to the high level of expression, incubation of the seedlings between 2 and 6 d after imbibition (DAI) with GUS staining solution was performed between 30 min and 1 h at 37°C. Two DAI, NADP-ME1::GUS expression was restricted to the radicle (Fig. 5a) and became more pronounced in the root tip by 4 DAI (Fig. 5b). Hypocotyl and cotyledons showed GUS expression by 5 DAI onward, while the expression tends to disappear from the root tissue. No GUS staining was evident in the primary leaves (Fig. 5c). At 2 DAI, GUS expression directed by the NADP-ME2 promoter was restricted to the proximal part of the radicle having root hairs (Fig. 5d). During the next days, strong staining expanded toward the meristematic region, except for the root tip (Fig. 5e). Strong expression was obtained in hypocotyls and cotyledons 6 DAI when the seedlings were incubated for more than 1 h in the staining solution. Primary leaves also showed GUS expression (Fig. 5f). GUS activity could not be detected in NADP-ME3::GUS lines at any stages of germination (Fig. 5, g and h). At later stages, GUS staining could be observed in stipules at the shoot apex and restricted to trichomes of the primary leaves (Fig. 5i). In the case of NADP-ME4::GUS lines, GUS expression was confined to the radicle 2 DAI (Fig. 5j) also approaching the meristematic region 3 DAI. At 4 DAI, the whole root was strongly stained and a low expression was also visible in the cotyledons when incubated in the staining solution for not longer than 1 h (Fig. 5k). By increasing the incubation time, strong expression was also seen in cotyledons. In contrast to the hypocotyls, the cotyledons displayed a strong GUS expression 7 DAI and this pattern was maintained with the appearance of true leaves (Fig. 5l).
Expression in Vegetative Organs NADP-ME1::GUS plants showed GUS expression only in some secondary roots, where it was confined to the stele (Fig. 6a) and excluded from the tips (data not shown). No GUS activity was detectable in leaves and stems throughout growth (data not shown). In NADP-ME2::GUS plants, reporter gene activity was found in all vegetative organs. Roots showed expression throughout all the tissues, except for root tips (Fig. 6b). Leaves exhibited GUS activity in all cell types, being particularly strong in the trichome basal cells and hydatodes (Fig. 6e). Stems showed strong GUS staining particularly in the vasculature (Fig. 6h). GUS expression was restricted to the trichomes and trichome basal cells of leaves and stems of NADP-ME3::GUS plants (Fig. 6, f and i). Interestingly, these plants showed expression of the NADP-ME3::GUS fusion in the stipules flanking the base of the inflorescence bract leaves (Fig. 6f) and the meristematic zone of developing lateral roots (Fig. 6c). NADP-ME4::GUS expression was found in all vegetative organs. Roots of these transgenic plants showed GUS staining in the stele, including the vascular tissue and the pericycle, mainly at emerging lateral roots and at root tips (Fig. 6d). In leaves and stems, GUS expression was also more intense in the vascular system (Fig. 6, g and j). NADP-ME4::GUS staining was found in guard cells of cotyledonous leaves, hypocotyls, and petioles (Fig. 6g). However, GUS activity was not obvious in guard cells of rosette and cauline leaves.
Expression in Reproductive Organs GUS activity staining of inflorescences indicated that NADP-ME1::GUS is not expressed in the floral organs (data not shown). NADP-ME2::GUS was expressed in sepals and filaments of developed flowers (Fig. 6k). In developing siliques, GUS staining was found at both ends, the stigmatic papillae and the abscission zone. The expression of NADP-ME3::GUS was found exclusively in pollen from the latest maturation stages up to its germination on the stigma (Fig. 6l). It is worth mentioning that, due to the high expression level of NADP-ME3, the inflorescences were incubated for only 3 h in the staining solution. The abscission zone of developing siliques also displayed GUS activity. NADP-ME4::GUS plants presented GUS staining in sepals, stigma, filaments, or pollen of opened flowers (Fig. 6m).
Several T-DNA mutants defective in the different AtNADP-ME genes could be identified in the SALK and SAIL collections. In all cases, homozygous lines were generated and confirmed using a PCR-based approach (data not shown). The position of the T-DNA insertion was determined by PCR amplification and sequencing of both ends of the insertion and of the flanking genomic DNA (Fig. 7A). Insertion line nadp-me1 presented a concatemer insertion in the orientation left border (LB)-LB in intron 15 (positions 2419–2432). In the case of nadp-me2, the insertion was localized to exon 5 (positions 983–1012), with an orientation right border (RB)-LB. Two additional lines of nadp-me2 were also analyzed. In the case of nadp-me2a and 2b, insertions with the orientation LB-RB in intron 15 (position 2904) and in exon 16 (positions 3141–3144), respectively, were obtained (data not shown). An insertion in intron 16 (positions 3161–3201) in the orientation LB-LB was identified in the line nadp-me3. Finally, T-DNAs arranged in tandem repeats with the orientation LB-LB inserted in intron 11 (positions 2041–2054) in nadp-me4 were confirmed. All lines showed no detectable expression of the corresponding genes as analyzed by RT-PCR using flower cDNA in the cases of nadp-me2, -me3, and -me4 and root cDNA in the case of nadp-me1 (Supplemental Fig. 1). Under greenhouse conditions, normal vegetative and reproductive development for all the single mutants was observed. To test for redundancy, homozygous double mutants were generated between the following pairs: nadp-me1 and nadp-me2, nadp-me1 and nadp-me4, nadp-me2 and nadp-me3, and nadp-me2 and nadp-me4. Also, homozygous triple mutants were generated by crossing the following homozygous lines: nadp-me1 x 2 with nadp-me2 x 4 and nadp-me2 x 3 with nadp-me2 x 4. None of the double- or triple-mutant combinations showed an obvious phenotype under normal growth conditions. A quadruple mutant still could not be isolated.
Contribution of Each Isoform to the NADP-ME Activity in Different Organs of Arabidopsis To correlate NADP-ME mRNA abundance and gene expression patterns with NADP-ME enzymatic activities, Arabidopsis ecotype Columbia (Col-0) and T-DNA insertion mutants were used to analyze the NADP-ME activity using different approaches. Protein extracts from the different lines were assayed for NADP-ME activity and analyzed by native PAGE electrophoresis. In addition, in situ NADP-ME activity staining of 4-week-old plants was performed. The distribution of NADP-ME activity obtained in different organs of Col-0 and T-DNA insertion mutants is summarized in Figure 7B. In the wild type, as well as in all the mutants except nadp-me2, roots represented the highest specific activities (Fig. 7B). The results obtained indicate that the three nadp-me2 (nadp-me2, -me2a, and -me2b) knockout mutants display a consistent decrease in NADP-ME activity in all organs tested. In Figure 7B, only the results for line nadp-me2 are shown, but the other two insertion lines presented the same behavior (data not shown). NADP-ME activity measurements in organs of nadp-me4 show a significant decrease of activity in flowers and roots. Controversially, the loss of the NADP-ME3 transcript, which is predominantly expressed in flowers, is accompanied by a significant increase of NADP-ME activity in leaves. Figure 8A shows results from native PAGE analyses followed by NADP-ME activity staining. In all tissues analyzed, a high molecular mass NADP-ME (>400 kD) can be detected in the wild type. This band may correspond to NADP-ME2 because this isoform is present in all organs (Fig. 2B) and the recombinant purified NADP-ME2 protein possesses this molecular mass as revealed by native PAGE (Fig. 3B). To a lesser extent, NADP-ME1 and -ME3 exhibiting the same molecular mass (Fig. 3B) may contribute to this active band. Two other additional active bands are present in different organs of the wild type, consistent with tetrameric and dimeric states of the enzyme, respectively. These two additional bands are more pronounced in flowers and may correspond to NADP-ME4, because the messenger for this isoform is present in all organs (Fig. 2B) and only the recombinant purified NADP-ME4 protein is present in dimeric and tetrameric states (Fig. 3B). In this way, the oligomeric states observed for the recombinant isoforms are also obtained from Arabidopsis crude extracts.
The high molecular mass active NADP-ME is present in nadp-me1, nadp-me3, and nadp-me4, but is absent in all organs of nadp-me2 (which is the mutant with the lowest NADP-ME activity in all the organs tested; Fig. 7B), except for flowers (Fig. 8A). This band may represent NADP-ME3, which is predominantly expressed in this organ (Fig. 6l). The lower molecular mass bands, probably due to NADP-ME4 (see above), are visible in all organs of nadp-me2 and may be responsible for the remaining NADP-ME activity measured. These bands are hardly detected in nadp-me4, but visible in both nadp-me1 and nadp-me3 (Fig. 8A). We also performed in situ NADP-ME activity staining of Col-0 and T-DNA insertion mutant seedlings. The results (Fig. 8B) demonstrate that nadp-me1, -me3, and -me4 still possess high NADP-ME activity levels, whereas NADP-ME activity is almost absent in nadp-me2 when compared to the negative control (Col-0 without malate). This result reinforces the idea that the cytosolic NADP-ME2 isoform contributes most to the major global NADP-ME activity, at least in roots and leaves.
Arabidopsis Contains Four NADP-ME Isoforms with Different Biochemical and Structural Properties
A comprehensive characterization of the entire NADP-ME family in the C3 dicot plant Arabidopsis is presented. The presence of both cytosolic and plastidic isoenzymes suggests that the coexistence of both kinds of NADP-MEs may be universal for all plant species, and both kinds of isoforms are also present in the monocot plant rice (Chi et al., 2004
Three putative cytosolic isoforms and one plastidic NADP-ME isoform are expressed in mature tissues of Arabidopsis. Because all three nadp-me2 are the only insertional mutants with strong decreased levels of NADP-ME activity, it can be suggested that NADP-ME2 is responsible for the major part of NADP-ME activity in mature tissues of Arabidopsis (Figs. 7B and 8B). This isoform, along with the plastidic counterpart (NADP-ME4), are the only isoforms showing a constitutive pattern of expression in Arabidopsis (Figs. 2B and 4–6
Different oligomeric states were observed for the recombinant purified AtNADP-ME isoforms by native gel electrophoresis (Fig. 3B). While the plastidic NADP-ME (NADP-ME4) assembles in equilibrium between dimers and tetramers, the other isoforms are present in a higher oligomeric state (Fig. 3B). In this way, NADP-ME4 resembles the oligomerization pattern observed for the recombinant maize plastidic photosynthetic (dimer or tetramer, depending on the pH; Detarsio et al., 2003
The kinetic characterization of AtNADP-ME isoforms revealed significant differences (Table I). The kcat obtained for NADP-ME2, NADP-ME3, and NADP-ME4 was very high compared to values previously reported for nonphotosynthetic enzymes (Drincovich et al., 2001
The low Km (malate) value of the plastidic isoform (NADP-ME4) in relation to values obtained for the cytosolic counterparts, especially NADP-ME1 and NADP-ME2 (Table I), could be explained by the observation that malate concentrations are normally higher in the cytosol than in plastids (Fridlyand et al., 1998
None of the homozygous T-DNA insertional mutants or the double or triple mutants produced showed a phenotype distinguishable from the wild type by growth under normal conditions. The lack of phenotypic defects may be explained by the fact that NADP-ME may be a redundant enzymatic function under normal growth conditions or, alternatively, there may be mutual redundancy of function between isoforms so that no individual isoform is essential. Moreover, we cannot rule out the possibility that there may be a compensatory change in activity of other isoforms when one is removed (Fig. 7B). On the other hand, the distinctive and specific patterns of GUS expression of the members of the AtNADP-ME family during the whole life cycle provide evidence for exclusive roles of each isoform in plant metabolism.
The patterns of expression of NADP-ME4 observed are consistent with the proposed role for NADP-ME in supporting fatty acid synthesis (Smith et al., 1992
Recently, C4 photosynthetic characteristics of cells of stems and petioles that surround the xylem and phloem were reported in tobacco and celery (Hibberd and Quick, 2002
In Arabidopsis, trichomes are specialized unicellular structures serving different functions, including the detoxification of cytotoxins and xenobiotics via glutathione conjugation (Gutiérrez-Alcalá et al., 2000
Pollen grains accumulate large amounts of intracellular oil bodies that act not only as energy reserves but also as a source for the rapid synthesis of membrane lipids after germination (Piffanelli et al., 1998
The characterization of Arabidopsis NADP-ME isoforms indicates different kinetic and biochemical properties and localization patterns for each member of the NADP-ME family. Some isoforms present particular characteristics that may be important to fulfill specific functions, while others are constitutively expressed but are localized to different subcellular compartments. The mutation of any member of the family or their combination did not result in any informative phenotype under greenhouse conditions. It could be possible that NADP-ME may be a redundant enzymatic function under these growth conditions. Alternatively, there may be compensatory changes in activities of other isoforms when one or more are removed. An extensive analysis of the T-DNA insertion mutants under different stress and growth conditions will be of particular interest to reveal the involvement of each NADP-ME isoform in plant metabolism.
Plant Lines and Growth Conditions
Following cold treatment (72 h at 4°C in the dark), Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0) and the T-DNA insertion lines were grown in a greenhouse under a 16-h-light/8-h-dark regime at 22°C. Alternatively, Arabidopsis seedlings were first sown on 0.5x Murashige and Skoog medium (Murashige and Skoog, 1962
Total RNA from roots and inflorescences of Arabidopsis was isolated from 100 mg of tissue using the TRIzol reagent (Gibco-BRL). RNA was converted into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen). Full-length cDNAs were amplified from inflorescence RNA, in the case of NADP-ME2 and NADP-ME3, and from root RNA in the case of NADP-ME1 and NADP-ME4, using platinum Pfx DNA polymerase (Invitrogen) and specific primers. Oligonucleotide primers were designed to introduce unique SalI and XhoI sites at the 5'- and 3'-ends, respectively. In order to express the mature NADP-ME4, specific primers containing the first codon downstream of the predicted transit peptide cleavage site (ChloroP V1.1 and TargetP Server v1.01; Emanuelsson et al., 1999
The pCR-Blunt II-TOPO plasmids containing the inserts of the different AtNADP-ME cDNAs were digested with SalI and XhoI and subcloned into the pET32 expression vector (Novagen). The constructs obtained were designed in such a way that, following enterokinase digestion of the chimeric proteins, only a few extra amino acid residues were introduced at the N terminus of the mature proteins.
In each pET32 vector containing the inserts of the different AtNADP-ME isoforms (pET-ME1, pET-ME2, pET-ME3, and pET-ME4), the NADP-ME is fused in frame to the His-tag in order to facilitate purification of the expressed fusion protein by a nickel-containing His-bind column (Novagen). The induction and purification of the fusion proteins were performed as previously described for the maize (Zea mays) photosynthetic and nonphotosynthetic NADP-ME (Detarsio et al., 2003
Different tissues (leaf, stem, flowers, and roots) of 6-week-old Arabidopsis wild-type and T-DNA insertion lines were ground in N2 and the resulting powder was suspended in 100 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 10% (v/v) glycerol, and 10 mM 2-mercaptoethanol in the presence of a protease inhibitor cocktail (Sigma). The homogenates were clarified by centrifugation and the supernatants were separated for activity measurements or subjected to electrophoresis.
NADP-ME activity was determined spectrophotometrically using a standard reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.5 mM NADP, and 10 mM L-malate in a final volume of 0.5 mL. The reaction was started by the addition of L-malate. Initial velocity studies were performed by varying the concentration of one of the substrates around its Km value while keeping the other substrate concentrations at saturating levels. All kinetic parameters were calculated at least by triplicate determinations and adjusted to nonlinear regression using free concentrations of all substrates (Detarsio et al., 2003
SDS-PAGE was performed in 10% (w/v) or 7.5% to 15% (w/v) linear gradient polyacrylamide gels according to Laemmli (1970) Native PAGE was performed using a 6% (w/v) acrylamide separating gel. Electrophoresis was run at 150 V at 10°C. Gels were assayed for NADP-ME activity by incubating the gel in a solution containing 50 mM Tris-HCl, pH 7.5, 10 mM L-malate, 10 mM MgCl2, 0.5 mM NADP, 35 µg/mL nitroblue tetrazolium, and 0.85 µg/mL phenazine methosulfate at 30°C.
To analyze the expression of the AtNADP-ME genes, total RNA from different organs (6-week-old Arabidopsis plants) was isolated and reversed transcribed as stated above. PCR reactions were conducted in a final volume of 10 µL using 0.5 µL of the transcribed product and Taq DNA polymerase (Qiagen). To achieve specific amplification products, one pair of primers was designed for each gene in a way that an intron was spanned in the genomic DNA. The pairs of primers used were as follows: NADP-ME1for (5'-ATGGAGAAAGTGACCAACTCAGA-3') and NADP-ME1rev (5'-TCGGATATTGTTCAACAGCCTCTTC-3'); NADP-ME2for (5'-ATGGGAAGTACTCCGACTGATTT-3') and NADP-ME2rev (5'-ACCCGGGTAGTTAACTAATGAGCATCTCT-3'); NADP-ME3for (5'-ATGGGCACCAATCAGACTCAGAT-3') and NADP-ME3rev (5'-ACCCGGGTATTGTAACGTGGATCACGCAT-3'); and NADP-ME4for (5'-ATGATCTCTCTCACTCCCTCGTT-3') and NADP-ME4rev (5'-ACCCGGGATCCTGAACTCCACCAGATACGGT-3'). As control, the Actin2 gene was amplified. The primers used were Actin2Sfor (5'-TGTACGCCAGTGGTCCTACAACC-3') and Actin2Brev (5'-GAAGCAAGAATGGAACCACCG-3'). Amplification conditions were as follows: 3-min denaturation at 94°C; 29 to 35 cycles at 94°C for 30 s, 53°C for 40 s, and 72°C for 30 s, followed by 5 min at 72°C. PCR products were resolved on a 1.5% (w/v) agarose gel.
To localize the NADP-ME expression in Arabidopsis, promoter regions of all four members of the AtNADP-ME family were amplified by PCR and cloned into the SmaI-HindIII or, alternatively, the SmaI-XbaI sites of the binary vector pGPTV-BAR that carries the GUS gene (Jefferson et al., 1987
The NADP-ME4::GFP construct contained the first 252 nucleotides of the NADP-ME4 coding region fused to the GFP coding sequence, flanked by the cauliflower mosaic virus 35S promoter and terminator sequences. The selected NADP-ME4 cDNA region was amplified using the primers NADP-ME4GW for (5'-CACCATGACTCTCTCACTCCCTCG-3') and NADP-ME4GWrev (5'-ATCCTGAACTCCACCAGATACGGT-3') and cloned into pENTR/D-TOPO (Invitrogen). GFP fusion constructs were made by subcloning the coding sequence into pGWB5 (for C-terminal GFP), a gateway-compatible binary vector designed for 35S promoter-driven expression of GFP fusion proteins (kindly provided by T. Nakagawa, Shimane University, Izumo, Japan). Cloning using gateway vectors was performed using reagents and protocols from Invitrogen.
Protoplasts were prepared from tobacco (Nicotiana tabacum) BY-2 cells and transiently transformed with polyethylene glycol, as described by Negrutiu et al., (1987)
SALK insertion lines 036898 (nadp-me1), 073818 (nadp-me2), 020607 (nadp-me2a), 139336 (nadp-me3), and 064163 (nadp-me4) and the insertion line SAIL_752_C09 (nadp-me2b) were obtained from the Nottingham Arabidopsis Stock Center (NASC; http://www.arabidopsis.info). The genotype of the lines was determined using a PCR-based approach. Basically, genomic DNA was isolated from individual plants and used as a template for PCR amplifications of wild-type and nadp-me alleles. The position of the T-DNA insertion sites into the nadp-me genes was verified by amplifying and sequencing the T-DNA flanking genomic DNA.
Four-week-old plants were fixed in 2% (w/v) paraformaldehyde and 1 mM dithiothreitol in phosphate-buffered saline, pH 7.0 (0.1% [w/v], Na2HPO4, 0.03% [w/v] NaH2PO4, and 0.9% [w/v] NaCl) at 4°C for 1 h and then rinsed overnight with water at 4°C (Sergeeva et al., 2004
We thank Esther Grube for help in transient GFP expression studies. M.F.D. and C.S.A. are members of the Researcher Career of CONICET, and M.C.G.W. is a fellow of the same institution and Rosario National University. Received May 19, 2005; returned for revision June 13, 2005; accepted June 13, 2005.
1 This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica (PICT 1–11604, Argentina), Fundación Antorchas (project no. 4248–63, Argentina), SeCyt-DAAD (DA/PA05–BI/016), and CONICET (PIP 3029) as well as the Deutsche Forschungsgemeinschaft (to V.G.M.). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.065953. * Corresponding author; e-mail candreo{at}fbioyf.unr.edu.ar; fax 54–341–4370044.
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-glucuronidase (GUS) fusions. Based on these results, possible novel roles for some members of the NADP-ME family in Arabidopsis are discussed. 
