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First published online January 25, 2008; 10.1104/pp.107.114975 Plant Physiology 146:1540-1552 (2008) © 2008 American Society of Plant Biologists Arabidopsis NAD-Malic Enzyme Functions As a Homodimer and Heterodimer and Has a Major Impact on Nocturnal Metabolism1,[W]Centro de Estudios Fotosintéticos y Bioquímicos, Universidad Nacional de Rosario, 2000 Rosario, Argentina (M.A.T., M.C.G.W., C.S.A., M.F.D.); and Botanisches Institut, Universität zu Köln, 50931 Cologne, Germany (H.F., U.-I.F., V.G.M.)
Although the nonphotosynthetic NAD-malic enzyme (NAD-ME) was assumed to play a central role in the metabolite flux through the tricarboxylic acid cycle, the knowledge on this enzyme is still limited. Here, we report on the identification and characterization of two genes encoding mitochondrial NAD-MEs from Arabidopsis (Arabidopsis thaliana), AtNAD-ME1 and AtNAD-ME2. The encoded proteins can be grouped into the two clades found in the plant NAD-ME phylogenetic tree. AtNAD-ME1 belongs to the clade that includes known  -subunits with molecular masses of approximately 65 kD, while AtNAD-ME2 clusters with the known β-subunits with molecular masses of approximately 58 kD. The separated recombinant proteins showed NAD-ME activity, presented comparable kinetic properties, and are dimers in their active conformation. Native electrophoresis coupled to denaturing electrophoresis revealed that in vivo AtNAD-ME forms a dimer of nonidentical subunits in Arabidopsis. Further support for this conclusion was obtained by reconstitution of the active heterodimer in vitro. The characterization of loss-of-function mutants for both AtNAD-MEs indicated that both proteins also exhibit enzymatic activity in vivo. Neither the single nor the double mutants showed a growth or developmental phenotype, suggesting that NAD-ME activity is not essential for normal autotrophic development. Nevertheless, metabolic profiling of plants completely lacking NAD-ME activity revealed differential patterns of modifications in light and dark periods and indicates a major role for NAD-MEs during nocturnal metabolism.
The enzymatic oxidation of L-malate to pyruvate and CO2 is catalyzed by two classes of malic enzymes (MEs) with the general requirement for divalent cations: NADP-dependent ME (NADP-ME; EC 1.1.1.40) and NAD-dependent ME (NAD-ME; EC 1.1.1.39 or 1.1.1.38, depending on the ability to decarboxylate oxaloacetate [OAA]). In plants, NADP-ME isoforms function in chloroplasts and the cytosol (Drincovich et al., 2001  ; Gerrard Wheeler et al., 2005), while NAD-ME isoforms are found in mitochondria (Winning et al., 1994). Photosynthetic NADP-ME isoforms are involved in metabolism in chloroplasts (C4 plants) or the cytosol (Crassulacean acid metabolism [CAM] plants; Drincovich et al., 2001), while plastidic nonphotosynthetic isoforms have been suggested to play a role in plant defense responses and lipid biosynthesis (Smith et al., 1992; Eastmond et al., 1997; Casati et al., 1999; Maurino et al., 2001). Several biological roles were proposed for the cytosolic NADP-ME isoforms, including participation in plant defense responses, lignin biosynthesis, and control of cytosolic pH (Martinoia and Rentsch, 1994).
In some C4 and CAM plants, NAD-MEs provide CO2 for the Calvin cycle during photosynthetic metabolism (Hatch and Kagawa, 1974
Nonplant NAD-MEs are typically homotetrameric proteins (Caldes et al., 1978 The genome of Arabidopsis (Arabidopsis thaliana) possesses two genes encoding putative NAD-MEs. Due to the assumed central role of NAD-ME in plant mitochondria, the aim of this work was to characterize the AtNAD-ME gene family at the biochemical and functional levels. Based on the biochemical characterization of recombinant proteins and metabolite profiles of loss-of-function mutants, novel properties for plant NAD-MEs are described.
Cloning and Sequence Analysis of AtNAD-ME1 and AtNAD-ME2 Two putative AtNAD-ME genes are present in the Arabidopsis genome, AtNAD-ME1 (At2g13560) and AtNAD-ME2 (At4g00570). The full-length cDNAs encoding AtNAD-ME1 and AtNAD-ME2 were cloned by reverse transcription (RT)-PCR and sequenced. The deduced proteins have molecular masses of 69.6 and 66.6 kD, respectively, share 63% identity at the amino acid level and are both predicted to contain a mitochondrial targeting peptide by four different prediction programs (ARAMEMNON, http://aramemnon.botanik.uni-koeln.de/).
A phylogenetic analysis based on an alignment of the available plant NAD-ME full-length protein sequences indicated that NAD-MEs are divided into two clades: (1) the
Specific Expression of AtNAD-ME Genes To study the tissue-specific expression of AtNAD-ME genes, quantitative real-time (qRT)-PCR experiments were performed. Transcripts for both AtNAD-ME1 and AtNAD-ME2 were detected in all the organs tested. In all cases, the transcript levels of AtNAD-ME1 were higher than that of AtNAD-ME2 (Fig. 2A ). The comparison of the abundance of each transcript relative to the expression in leaves indicated that both genes have the same relative level of expression in all mature organs (Fig. 2B). The expression was similar in leaves and stems (100%), while the expression in flowers and roots accounted for 60% and 4% of the level in leaves, respectively (Fig. 2B).
To investigate more precisely the organ- and tissue-specific expression of both AtNAD-ME genes, transgenic Arabidopsis plants expressing the GUS reporter gene driven by the AtNAD-ME promoters were generated and analyzed throughout development. About eight transgenic AtNAD-ME::GUS lines were analyzed in detail, most of them showing a similar tissue-specific pattern of AtNAD-ME1 and AtNAD-ME2 expression. The pattern of GUS activity was very similar for the AtNAD-ME1::GUS and AtNAD-ME2::GUS plants (Fig. 2C). In both cases, GUS expression could be observed 2 d after imbibition (DAI) in the cotyledons, hypocotyls, and root tip (data not shown). At 3 DAI, the roots were completely stained (Fig. 2C, a and k) and in the case of AtNAD-ME1-promoter::GUS plants, the root tip was highly stained (Fig. 2C, a). A high expression was observed in trichomes and trichome basal cells, especially in AtNAD-ME1::GUS plants (Fig. 2C, b and l). At 5 DAI, both root tips were highly stained, and expression in all seedling tissues was maintained (Fig. 2C, c and ll). At 12 DAI, GUS expression was very low in the new leaves but became higher with maturation (Fig. 2C, d, e, m, and n). At all developmental stages, the expression in leaves was observed in the mesophyll and the cells that surround the vascular bundles (bundle sheet cells; Supplemental Fig. S1). Stems (Fig. 2C, f and o) and roots (Fig. 2C, g, h, p, and q) presented high expression in all tissues in both AtNAD-ME::GUS plants. It is interesting to note that longitudinal sections of stems revealed a high level of activity of both promoters around the vascular system (Fig. 2C, f and o). In the reproductive organs of both lines, GUS expression was detected in the apical part of the gynoecium, stigmatic papillae, the filaments, and sepals (Fig. 2C, i and r). In developing siliques, expression was high in the apical part and the abscission zone (Fig. 2C, i, j, r, and s). It is worth mentioning that the GUS expression driven by the AtNAD-ME1-promoter was always stronger than by AtNAD-ME2-promoter in all lines tested. It should also be noted that the observations described above are consistent with AtGenExpress data from the Genevestigator microarray database (Zimmermann et al., 2004 ; http://www.genevestigator.ethz.ch/).
To assess whether the two predicted AtNAD-MEs are enzymatically active proteins, AtNAD-ME1 and AtNAD-ME2 cDNAs were cloned and expressed as recombinant proteins. The prediction of the length of the mitochondrial targeting sequences was performed by sequence comparison with potato NAD-ME and the assistance of prediction programs (ARAMEMNON, http://aramemnon.botanik.uni-koeln.de/). After eliminating mitochondrial targeting sequences of 38 and 31 amino acid residues for AtNAD-ME1 and AtNAD-ME2, respectively, the mature proteins were expressed in Escherichia coli. Following induction of the expression by isopropyl-β-thiogalactopyranoside or lactose, proteins with the expected molecular masses of 80 and 76 kD (AtNAD-ME1 and AtNAD-ME2, respectively) were purified by affinity chromatography (Fig. 3A
). After enterokinase digestion to remove the His tag used for purification, products of 63 and 58 kD were obtained for AtNAD-ME1 and AtNAD-ME2, respectively (Fig. 3A). Both recombinant proteins were recognized by the anti-A. hypochondriacus
Interestingly, recombinant purified AtNAD-ME1 and AtNAD-ME2 showed both enzymatic activities and could thus be characterized with respect to their kinetic properties (Table I ). Both isoforms displayed similar kcat values, with AtNAD-ME2 having a 1.3-fold higher kcat value than AtNAD-ME1 (Table I). Comparing the apparent Km values for NAD and malate, AtNAD-ME1 and AtNAD-ME2 exhibited very similar affinities toward both compounds, while AtNAD-ME2 presented the highest catalytic efficiency (kcat/Km; Table I). It is worth mentioning that the kinetic behavior obtained for AtNAD-ME1 with respect to both NAD and malate was nonhyperbolic, presenting some kind of sigmoidicity and thus probable cooperative binding of the ligand in both cases (Table I). The pH optimum for both isoforms was very similar, at about pH 6.5 (Table I). Neither AtNAD-ME1 nor AtNAD-ME2 was able to decarboxylate OAA, even when using high protein concentrations for the assay (data not shown). Thus, both AtNAD-MEs clearly belong to the EC 1.1.1.39 subtype.
Native electrophoresis of the purified recombinant AtNAD-MEs was analyzed by activity staining and western blot (Fig. 3, B and C). An active band compatible with a dimeric oligomeric state for the recombinant AtNAD-ME2 was detected by activity staining assays (Fig. 3B). This active band also reacted with the anti-A. hypochondriacus -NAD-ME-antibody (Fig. 3C). Interestingly, the recombinant AtNAD-ME1 could not be detected by activity staining (even loading up to 40 milliunits of the purified enzyme), although a band with a higher mobility than that of AtNAD-ME2 was obtained by western blot using the anti-A. hypochondriacus -NAD-ME- antibody (Fig. 3C). As the purified AtNAD-ME1 exhibited enzymatic activity in solution, it is possible that the protein lost its activity during electrophoresis. Size-exclusion chromatography was used to estimate the native molecular masses for both recombinant AtNAD-MEs. The calculated molecular masses for the purified AtNAD-ME1 and AtNAD-ME2 proteins were 120.0 ± 6 and 117.5 ± 6.5 kD, respectively. Thus, although AtNAD-ME1 and AtNAD-ME presented different mobilities by native electrophoresis, probably due to differences in the charge, both proteins obviously assembled as dimers in solution.
NAD-ME activity was measured in different organs of mature Arabidopsis plants. Crude extracts from roots showed the highest activity expressed on the basis of total protein concentration (0.038 ± 0.03 units/mg), while leaves, stems, and flowers displayed between 58% and 65% of the activity measured in roots (Fig. 5C
). When extracts of isolated mitochondria from each organ were analyzed by SDS-PAGE followed by western-blot analysis, two well-separated bands could be detected using the anti-A. hypochondriacus
On the other hand, native PAGE of mitochondrial extracts from different Arabidopsis organs analyzed by western blot showed a band of approximately 120 kD using both specific antibodies directed against AtNAD-ME1 or AtNAD-ME2, while the recombinant proteins differ in electrophoretic mobilities and show specific reactions against their own antibodies (Fig. 4D). Moreover, a second immunoreactive band with the same mobility as the recombinant protein was detected in the mitochondrial extracts (Fig. 4D), indicating the possible existence of homodimers in vivo. The interaction of AtNAD-ME1 and AtNAD-ME2 observed in mitochondrial extracts was further tested in vitro using recombinant AtNAD-ME. Purified recombinant AtNAD-ME1 and AtNAD-ME2 were mixed in an equimolar ratio and subsequently analyzed by native PAGE. As shown in Figure 3, B and C, the mixture has active and immunoreactive bands as the isolated recombinant proteins, and, additionally, it has a band with similar mobility to that observed in the mitochondrial extracts. Taken together, these results revealed that the separated AtNAD-ME1 and AtNAD-ME2 assemble as active dimers and associate to form a heterodimer in vitro and in vivo.
Arabidopsis insertion mutants that contained T-DNA elements inserted in the AtNAD-ME1 and AtNAD-ME2 genes were isolated from the Sail-lines (Fig. 5A). Homozygous lines for each mutant were confirmed by PCR and designated nad-me1.1 (Sail-374-A02) and nad-me2.1 (Sail-291-C05), respectively. The sites of the insertions were analyzed by sequencing the PCR products obtained after amplifying both ends of the T-DNA insertion and the flanking genomic DNA. The knockout line nad-me1.1 had an insertion in exon 4 (at position +833 bp), and in line nad-me2.1 the insertion was localized in exon 5 (at position +1,030 bp). The mutant lines showed no expression of the corresponding genes as analyzed by RT-PCR (Fig. 5B), and the absence of the corresponding protein was confirmed by western blot using specific antibodies (data not shown). A second allelic mutant for each gene (Sail-374-A02, nad-me1.2 and Salk-131720, nad-me2.2) was also isolated and characterized in parallel, but the lines were not included in this work. As growth and development of all the single mutants analyzed did not show visual differences with respect to the wild type, homozygous double mutants were generated between nad-me.1.1 and nad-me2.1 by crossing. The absence of transcripts and proteins for both AtNAD-MEs in the double mutants was confirmed by RT-PCR and western blot (Fig. 5B; data not shown). Figure 5C shows residual NAD-ME activities measured in different organs from nad-me1.1 and nad-me2.1. The results indicated that in vivo, both AtNAD-ME1 and AtNAD-ME2 exhibit enzymatic activity and that AtNAD-ME2 possesses the highest specific activity, results that correlate well with those obtained for the recombinant proteins. The double mutant did not show any NAD-ME activity, indicating that AtNAD-ME1 and AtNAD-ME2 are solely responsible for the NAD-ME activities measured in crude extracts. Native PAGE conducted with mitochondria extracted from both single mutants showed an immunoreactive band corresponding to the remaining functional AtNAD-ME protein, while in the double knockout extracts, no immunoreactive band was detected (data not shown). It is worth mentioning that transcript levels of the remaining intact AtNAD-ME gene in the organs of each single mutant did not show significant difference relative to the expression observed in the wild-type organs when analyzed by qRT-PCR (data not shown). The germination, development, vegetative growth, and flowering time of the double knockouts were very similar to that of the wild type and the single knockout mutants when grown either at moderate (100 µmol m–2 s–1) or high (500 µmol m–2 s–1) light intensities. The data obtained indicated that there are no statistical differences in rosette diameter or dry weight between the wild type and homozygous mutants grown in both conditions (Table II ; data not shown). Moreover, the photosynthetic parameters maximum quantum efficiency of PSII and electron transport rate (ETR; Table II) and the qP and qN values (data not shown) indicated no differences between the knockout mutants and the wild type.
AtNAD-ME Is More Active during the Night Period, and Mutant Lines Completely Lacking NAD-ME Activity Display Altered Steady-State Levels of Sugars and Amino Acids
To gain further information about the extent of physiological disturbances generated by the lack of total NAD-ME activity, metabolite profiling analyses using gas chromatography-mass spectrometry (GC-MS; Fahnenstich et al., 2007
Due to the differences found in the metabolic profile in the double loss-of-function mutant, the total NAD-ME activity, NAD-ME protein amount and the expression levels of both genes were analyzed at the end of the light and night periods in the wild type. Leaf crude extracts contained about 20% higher NAD-ME specific activities at the end of the night period than at the end of the day period (Fig. 6A ). In line with this result, western-blot analysis of these extracts showed that both AtNAD-ME1 and AtNAD-ME2 were more abundant during the night period (Fig. 6B). Quantification of the amount of immunoreactive protein of three biological replicates indicated that AtNAD-ME1 and AtNAD-ME2 were enhanced approximately 3- and 2.5-fold in the night period with respect to the day period, respectively. Moreover, the expression of both genes, evaluated by qRT-PCR, was also enhanced by the end of the night (Fig. 6C). Taken together, Arabidopsis leaf extracts presented higher NAD-ME activity during the night period as a result of protein accumulation due to enhanced gene expression.
Arabidopsis Possesses a Heterodimeric NAD-ME, and Both Subunits Exhibit Enzymatic Activities in Vitro and in Vivo
AtNAD-ME1 and AtNAD-ME2 show homologies to the
A heteromeric composition of plant NAD-MEs has been reported for some species (Grover and Wedding, 1982
The kinetic analyses performed showed that AtNAD-ME2 displayed a slightly higher specific activity in comparison to AtNAD-ME1 (Table I). Nevertheless, the pH optimum and the affinity for NAD and malate were almost identical for both proteins, and this data is in agreement with previous reports on plant NAD-ME (Artus and Edwards, 1985 Finally, in accordance to the universal role in plant mitochondria, the AtNAD-ME1 and AtNAD-ME2 genes have very similar levels of expression in all mature organs. It is worth mentioning that the level of expression of the AtNAD-ME1 transcript was higher than that of AtNAD-ME2 in all the organs analyzed; nevertheless, this does not necessarily imply a higher level of AtNAD-ME1 protein.
As NAD-ME was assumed to play a central role in the management of flux through the TCA cycle by providing a means of generating acetyl-CoA, and thus, ATP and carbon skeletons (Grover et al., 1981
On the contrary, at the end of night, the levels of amino acids derived from OAA and 2-oxoglutarate were increased in the mutants completely lacking NAD-ME activity, particularly Asn and Gln. At the same time, these plants showed enhanced levels of the TCA cycle intermediates 2-oxoglutarate and succinate compared to the wild type. Interestingly, the levels of amino acids derived from pyruvate, e.g. Ala, Val, and Ile, were invariable in the mutant. In Arabidopsis, the malate level increases during the day, and this organic acid is accumulated in the vacuole until metabolic demands are sensed (Gout et al., 1993
It was a matter of debate whether or not mitochondrial NAD-ME could compensate for limited capacity for pyruvate transport across the mitochondrial membrane by providing the TCA cycle with pyruvate under conditions of high-energy demands (Day and Hanson, 1977 ; Brailsford et al., 1986; Hill et al., 1994). It would thus be interesting to challenge the nad-me double knockout mutant to different conditions in order to prove this hypothesis.
On the other hand, high activities of NAD-ME and NADP-ME were reported in cells around the vascular bundles in tobacco (Hibberd and Quick, 2002
Isolation of T-DNA Insertion Lines and Plant Growth Conditions The T-DNA insertion lines Sail-374-A02 (nad-me1.1) and Sail-291-C05 (nad-me2.1) were obtained from the Nottingham Arabidopsis Stock Center (http://www.arabidopsis.info/). The genotype of the lines was determined using genomic DNA of individual plants as template for PCR amplifications of the wild-type and nad-me alleles. The primers used to amplify the wild-type alleles were as follows: NAD-ME1wtF (5'-ACGATGACGGAGAGAATCGT-3') and NAD-ME1wtR (5'-ATGTTCAATGATGATGTCCAG-3'), and NAD-ME2wtF (5'-GACCTGTGTACAGCAATGTGATCG-3') and NAD-ME2wtR (5'-GGTCTTGTCACCACGGAGAGGACA-3'). To amplify the nad-me alleles, the primers NAD-ME1wtF and NAD-ME2wtF were combined separately in a PCR with the primer SailLB (5'-TAGCATCTGAATTTCATAACCAATCTCGATACAC-3'). The location of the inserts was verified by amplifying and sequencing the T-DNA flanking genomic DNA. Seeds of Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 and the transformant lines were placed on soil and kept in darkness for 4 d at 4°C to synchronize germination. After 2 weeks, the seedlings were transferred to pots (one per pot) containing three parts of soil (Gebr. Patzer KG) and one part of vermiculite (Basalt Feuerfest). Plants were grown under a 16-/8-h photoperiod at 100 or, alternatively, at 500 µmol m–2 s–1 and 21°C/18°C (day/night) temperatures and 65% relative humidity in a controlled growth cabinet. Alternatively, Arabidopsis seeds were sterilized and sown in Murashige and Skoog agar plates containing 1% Suc.
Arabidopsis full-length cDNAs encoding NAD-ME1 and NAD-ME2 were amplified by RT-PCR using RNA extracted from leaves and the TRizol reagent (Gibco-BRL). Amplification was conducted using SuperScript II reverse transcriptase (Invitrogen) and specific primers. In the case of AtNAD-ME1, the oligonucleotide pair NAD-ME1GWF (5'-CACCATGGGAATAGCCAATAAGCTCCGGCT-3) and NAD-ME1GWF (5'-GAGTACCCGACTTTGGTCTACAAGGATGAC-3') was used. AtNAD-ME2 was amplified with the primer pair NAD-ME2GWF (5'-CACCATGTGGAAGAACATTGCTGGGTTGTC-3') and NAD-ME2GWR (5'-CCTGTTTACAGCCCTCTCGTTCACGAGAAA-3'). The PCR products were cloned into pENTR/D-TOPO (Invitrogen) and completely sequenced.
To amplify the cDNA fragments corresponding to the mature AtNAD-MEs, a PCR reaction was conducted using as template the full-length cDNAs cloned as described above and the following primer pairs: NAD-ME1F (5'-GGATCCCCCACCATCGTTCATAAA-3') and NAD-ME1R (5'-GTCTACAAGGATGACTAAGTCGAC-3'), and NAD-ME2F (5'-GGATCCTGCATCGTCCACAAGCGT-3') and NAD-ME2R (5'-ACGCTTGTGGACGATGCAGGATCC-3'). The primers were designed to introduce unique BamHI and SalI sites at the 5' and 3' ends, respectively, to facilitate the subcloning into the pET32 expression vector. In each pET32 vector containing the inserts of AtNAD-ME1 and AtNAD-ME2 (pET-NAD-ME1 and pET-NAD-ME2), the NAD-MEs are fused in-frame to a His tag 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 Arabidopsis NADP-ME isoforms (Gerrard Wheeler et al., 2005
Molecular masses of recombinant native AtNAD-ME1 and AtNAD-ME2 were evaluated by gel filtration chromatography on a FPLC system using a Superdex 200 10/300 GL column (Amersham Biosciences). The column was equilibrated with 25 mM Tris-HCl, pH 7.5, or with 50 mM MES-NaOH, pH 6.5, and calibrated using molecular mass standards. The sample and the standards were applied separately in a final volume of 50 µL at a constant flow rate of 0.5 mL/min.
Polyclonal antibodies against recombinant AtNAD-ME1 and AtNAD-ME2 were obtained by immunization of rabbits with 200 µg of the purified proteins in four subcutaneous injections of 50 µg at 15-d intervals. The antibodies against the recombinant AtNAD-MEs were further purified from the crude antiserum (Plaxton, 1989
Different Arabidopsis organs (leaf, stem, flowers, and roots) of 6-week-old wild-type and T-DNA insertion lines were ground in N2, and the resulting powder was suspended in buffer B (50 mM MES-NaOH pH 6.5, 5 mM MnCl2, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.05% Triton X-100, 20% glycerol, and 1 mm phenylmethylsulfonyl fluoride). The homogenates were clarified by centrifugation. The supernatants were desalted using a Sephadex G-50 column equilibrated with buffer A and separated for activity measurements or subjected to electrophoresis.
Mitochondria from leaves, stems, roots, and flowers were prepared by a modification of the method previously described by Keech et al. (2005)
NAD-ME activity in crude extracts (whole plant tissues or isolated mitochondria) was measured spectrophotometrically using a standard reaction mixture containing 50 mM MES-NaOH, pH 6.5, 4 mM NAD, 10 mM malate, 5 mM dithiothreitol; 10 mM MnCl2, and 10 units of MDH. There was a rapid but small increase of the A340 as the reaction catalyzed by the MDH reached the equilibrium. With the assay system specified above, the subsequent steady increase of A340 was attributable to the decarboxylation of L-malate by the NAD-ME (Chapman and Hatch, 1977
In the case of purified recombinant AtNAD-ME, enzymatic activity was determined spectrophotometrically using a standard reaction mixture containing 50 mM HEPES, pH 6.4 or 6.6 (for AtNAD-ME1 and AtNAD-ME2, respectively), 10 mM MnCl2, 4 mM NAD, 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 substrates concentrations at saturating levels. All kinetic parameters were calculated at least by triplicate determinations and adjusted to nonlineal regression using free concentrations of all substrates. OAA decarboxylation was monitored by measuring the OAA disappearance at 260 nm (
One unit is defined as the amount of enzyme that catalyzes the formation of 1 µmol of NADH min–1 under the specified conditions. Protein concentration was determined by the method of Sedmak and Grossberg (1977)
Denaturing PAGE (SDS-PAGE) was performed in 10% (w/v) or 7.5–15% (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 NAD-ME activity by incubating the gel in a solution containing 50 mM HEPES, pH 6.5, 60 mM malate, 4 mM NAD, 10 mM MnCl2, 35 µg/mL nitroblue tetrazolium, and 10 µg/mL phenazine methosulfate at 30°C. Alternatively, native gels were electroblotted onto a nitrocellulose membrane and subjected to western-blot analysis.
To evaluate the expression of the AtNAD-ME genes in the T-DNA insertional mutants, total RNA from leaves were isolated from 100 mg tissue using the TRIzol reagent (Gibco-BRL). RNA was converted into first-strand cDNA using the SuperScriptII Reverse Transcriptase (Invitrogen). PCR reactions were conducted in a final volume of 10 µL using 1 µL of the transcribed product and Taq DNA polymerase (Qiagen). The pairs of primers used were NAD-ME1wtF and NAD-ME1wtR and NAD-ME2wtF and NAD-ME2wtR. Amplification conditions were as follows: 3 min denaturation at 94°C; 35 cycles at 94°C for 30 s, 53 to 55°C for 40 s and 72°C for 30 s, followed by 5 min at 72°C. As control, the actin2 gene was amplified by 28 cycles and the following primers were used: actin2-F (5'-TAACTCTCCCGCTATGTATGTCGC-3') and actin2-R (5'-GTACGGTAACATTGTGCTCAGTGG-3').
Relative gene expression was determined by performing qRT-PCR in an iCycler iQ detection system and the Optical System Software version 3.0a (Bio-Rad), using the intercalation dye SYBRGreen I (Invitrogen) as a fluorescent reporter, with 2.5 mM MgCl2, 0.5 µM of each primer, and 0.04 units/µL GoTaq (Promega). A 2-fold dilution of cDNA was used as template. PCR controls were performed in the absence of added reverse transcriptase to ensure that RNA samples were free of DNA contamination. Cycling parameters were as follows: initial denaturation at 94°C for 2 min; 40 cycles of 96°C for 10 s, and 56°C for 15 s; 72°C for 1 min; and 72°C for 10 min. Melting curves for each PCR reaction were determined by measuring the decrease of fluorescence with increasing temperature (from 65°C to 98°C). The specificity of the PCR reactions was confirmed by melting curve analysis using the software as well as by agarose gel electrophoresis of the products. Relative gene expression was calculated using the comparative 2–
For the generation of promoter-GUS constructs, fragments containing a 1.7-kb promoter region upstream the ATG start codon, the first exon and intron and part of the second exon of both AtNAD-ME genes were amplified by PCR from genomic DNA. The following primer pairs were used: pNAD-ME1GWF (5'-CACCTCGAGAGTTCTTAGCTAAACAATCT-3') and pNAD-ME1GWR (5'-GGGACTGCGTTTACGATGACGGAGAGA-3'), and pNAD-ME2GWF (5'-CACCATGGGTTGGAGCAGATGGATT-3') and pNAD-ME2GWR (5'-ATACGAGGCTTGCTTCCTCCTCGT-3'). The amplified products were sequenced and cloned into pGWB3, a gateway-compatible binary vector that carries the GUS gene and the kanamycin resistance gene (provided by T. Nakagawa, Shimane University, Izumo, Japan). The resulting constructs were introduced into Arabidopsis by A. tumefaciens transformation using the vacuum infiltration method. Transgenic lines were selected on Murashige and Skoog plates containing kanamycin (50 µg/mL). The histochemical localization of GUS activity was conducted as described by Maurino et al. (2006)
Chlorophyll fluorescence measurements were performed with a PAM-2000 pulse amplitude modulated chlorophyll fluorometer (Walz). At the start of each measurement, a plant was dark adapted for 10 min. Basal fluorescence was measured with modulated weak red light and maximal fluorescence was induced with a saturating white light pulse (5,000 µmol m–2 s–1; duration 0.8 s).
Whole rosettes were harvested at the end of the light and dark periods and transferred into liquid nitrogen in less than 10 s. At least 8 rosettes were combined per sample. The leaves were homogenized using liquid nitrogen and stored at –80°C until use. The extraction and GC-MS analysis was conducted as described by Fahnenstich et al. (2007)
Significance was determined according to the Student's t test using Excel software (Microsoft).
The following materials are available in the online version of this article.
We thank Andreas Weber for helpful discussions and Ulrike Hebbeker and Claudia Nothelle for technical assistance. Received December 13, 2007; accepted January 23, 2008; published January 25, 2008.
1 This work was supported by the Deutsche Forschungsgemeinschaft (to V.G.M.), and by CONICET and Agencia Nacional de Promoción de Actividades Científicas y Tecnológicas (to M.F.D. and C.S.A.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Verónica G. Maurino (v.maurino{at}uni-koeln.de).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.114975 * Corresponding author; e-mail v.maurino{at}uni-koeln.de.
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260 nm = 850 M–1 cm–1) in an assay medium containing 50 mM MES-NaOH, pH 5.5, 1 mM OAA, and 10 mM MnCl2. 
