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First published online November 20, 2003; 10.1104/pp.103.029363 Plant Physiology 133:1968-1978 (2003) © 2003 American Society of Plant Biologists Identification of AtNDI1, an Internal Non-Phosphorylating NAD(P)H Dehydrogenase in Arabidopsis Mitochondria1School of Biological Sciences, Flinders University of South Australia, G.P.O. Box 2100, Adelaide, South Australia 5001, Australia (C.S.M., R.J.C.-J., J.T.W., K.L.S.); Department of Biochemistry and Biophysics, Stockholm University, Stockholm S106 91, Sweden (C.R.); and Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia (J.W., D.A.D.)
Plant mitochondria contain non-phosphorylating NAD(P)H dehydrogenases (DHs) that are not found in animal mitochondria. The physiological function, substrate specificity, and location of enzymes within this family have yet to be conclusively determined. We have linked genome sequence information to protein and biochemical data to identify that At1g07180 (SwissProt Q8GWA1) from the Arabidopsis Genome Initiative database encodes AtNDI1, an internal NAD(P)H DH in Arabidopsis mitochondria. Three lines of evidence are presented: (a) The predicted protein sequence of AtNDI1 has high homology with other designated NAD(P)H DHs from microorganisms, (b) the capacity for matrix NAD(P)H oxidation via the rotenone-insensitive pathway is significantly reduced in the Atndi1 mutant plant line, and (c) the in vitro translation product of AtNDI1 is imported into isolated mitochondria and located on the inside of the inner membrane.
The plant mitochondrial inner membrane contains a branched electron transport pathway that includes multiple enzymes for the oxidation of both cytosolic and matrix NAD(P)H. A feature of the plant respiratory chain is the presence of alternative enzymes that are not linked to proton translocation and, hence, are non-phosphorylating. These include the alternative oxidase (AOX), which provides an alternative route for electrons from the ubiquinone pool to oxygen, and four non-proton pumping NAD(P)H dehydrogenases (DHs), identified by their insensitivity to the complex I inhibitor rotenone (Mackenzie and McIntosh, 1999  ; Møller, 2001). These alternative enzymes have the potential to catalyze wasteful respiration but can also decrease the production of reactive oxygen species in the respiratory chain (Møller, 2001). The synthesis and activity of AOX are strictly controlled in most plant tissues (Vanlerberghe and McIntosh, 1997), but much less is known about the alternative NAD(P)H DHs.
Yeast (Saccharomyces cerevisiae), which lacks Complex I, has at least three non-phosphorylating NAD(P)H DHs in its respiratory chain; ScNDE1, ScNDE2 (external facing), and ScNDI1 (internal facing). These have been well characterized (Marres et al., 1991
Recently, two putative NAD(P)H DHs have been identified from potato (Solanum tuberosum; Rasmusson et al., 1999 Advances in our understanding of the significance of the non-phosphorylating NAD(P)H DH family in plant mitochondria has been limited by the lack of a clear link between genomic knowledge and biochemical data. This paper describes the identification of an NAD(P)H DH gene by characterization of the mitochondrial phenotype of a publicly available Arabidopsis T-DNA insertion mutant. For the first time to our knowledge, molecular and functional evidence for the identity and location of an Arabidopsis non-phosphorylating NAD(P)H DH is presented. Our understanding of the functions, location, and substrate specificity of other plant non-phosphorylating enzymes, based on sequence analysis is discussed, and a standard nomenclature for identified plant genes and gene products that have been identified functionally is proposed.
Sequence Analysis
Six putative NAD(P)H DH sequences (AT1g07180 [AtNDI1], AT2g29990, AT2g20800, AT4g05020, AT4g28220, and AT4g21490) were identified from the Arabidopsis genome database, based on sequence similarity with the yeast genes ScNDE1, ScNDE2, and ScNDI1 (Table I; Figs. 1 and 2). With the exception of AT4g21490, all of the putative translated proteins from these sequences were predicted to be mitochondrially located by at least one of the public subcellular prediction programs: Psort, Predotar, and Target P (Table II). The fact that five of the six putative NAD(P)H DHs are predicted to be mitochondrial by two of the more commonly used prediction programs suggests a mitochondrial location. The poor prediction of targeting signal length by TargetP may be because at least some of these proteins are located on the intermembrane space side of the inner membrane and, thus, are not likely to contain a typical processing site for the matrix located mitochondrial processing peptidase. Thus, the predicted length of the targeting signal may not be reliable. Even though AT4g21490 was not predicted to target to a mitochondrial location, it was retained due to its high similarity to the characterized NAD(P)H DH sequences (Table I). The highest similarity was seen between AtNDI1 and AT2g29990, with 82% amino acid residue identity. The plant protein sequences separate into two groups that either show a high similarity to the previously characterized potato StNDA or StNDB sequences (Rasmusson et al., 1999
When aligned with the previously characterized proteins ScNDE1, ScNDE2, and ScNDI1 of yeast, NcNDE1 of N. crassa and with StNDB and StNDA of potato, the Arabidopsis sequences fall into two clear groups, based both on the presence of common and unique functional motifs within the ScNDE-like and ScNDI-like proteins and similarity to StNDB (Fig. 1) or StNDA (Fig. 2).
Both the NDA-like and NDB-like NAD(P)H DH sequences contain two dinucleotide fold motifs, characterized by the core GXGXXG signature (Wierenga et al., 1986
It is interesting to note that the predicted NDB-like enzymes are generally longer in length than the NDA-like enzymes (Table I; Figs. 1 and 2). This is due to an insertion of additional sequence that is present toward the C-terminal end of NcNDE1, three of the four Arabidopsis sequences, and the potato NDB protein (Fig. 1). This "insert" region contains two motifs that have homology to Ca2+-binding EF hand motif (Marsden et al., 1990
The Arabidopsis T-DNA database of insertion mutants (Sessions et al., 2002 Genomic PCR was carried out using an insert-specific primer (T-DNA-SP) in combination with a gene-specific primer (ATx-GSP) to confirm the presence or absence of the insert in the genes of interest. To determine whether the NDA-like genes (AT2g29990 and AtNDI1) were being expressed, reverse transcriptase (RT)-PCR was performed using the gene-specific 5'- and 3'-untranslated region (UTR) primers (At1 and At6, Table III). All amplifications were carried out in triplicate using DNA and RNA isolated from the separate Arabidopsis shaking cultures used for mitochondrial extraction and biochemical analyses. A schematic representation of the AtNDI1 gene (including introns) indicates the location of primers used with respect to the T-DNA insertion site (Fig. 3A). In the genomic PCR, the Atndi1 shaking seedling cultures gave no amplification product using T-DNA-SP in combination with AT1-GSP (data not shown). However, products of expected size were amplified in the Atndi1 T-DNA mutant replicates using T-DNA-SP in combination with AT6-GSP (Fig. 3B). This indicates that the T-DNA insert is in the AtNDI1 gene rather than At2g29990. Figure 3C shows the amplification of AT2g29990 cDNA in each of the replicate Atndi1 mutants and in wt samples (sequence identity confirmed by sequencing). In contrast, RT-PCR using the AtNDI1 gene-specific 5'-and 3'-UTR primers failed to amplify any cDNA product from the Atndi1 mutant RNA but amplified the full-length cDNA from wt (Fig. 3D).
From these results, we concluded that the mutant plants contain an insert in AtNDI1 that has completely suppressed the transcription of this gene, whereas the highly homologous At2g29990 gene is still transcribed. As a consequence, it was assumed that changes in the biochemical properties of mutant compared with wt mitochondria are attributable to disruption of AtNDI1. When wt and transgenic plants were grown under identical conditions, there was no observable morphological phenotype. The plants in shaking culture yielded approximately the same amount of tissue (fresh weight 20-30 g), shoot to root ratios were not significantly different, and overall appearance was similar (data not shown). This is not altogether surprising given that the transcription of only one of the two putative internal NAD(P)H DH genes is disrupted in the T-DNA insertion mutant plants.
Mitochondria were isolated from mutant and wt plants grown in shaking culture, and their electron transport capacity was measured. The assays were designed to measure the internal, external, and outer membrane NAD(P)H DH capacities separately in intact mitochondria. This was achieved by the selective use of FeCN as an artificial electron acceptor in conjunction with inhibitors of different parts of respiratory electron transport chain. Conditions were chosen to favor the production of NAD(P)H via malate DH and malic enzyme within the matrix (for details, see "Materials and Methods"). In all cases, enzyme activities are expressed as a ratio of the succinate DH activity for that mitochondrial preparation to compensate for any variation in the respiratory activity of individual mitochondrial extractions. Total matrix malate-FeCN reductase activity ratio was approximate 40% lower in the mutant plants compared with wt (Fig. 4). This was due entirely to a decrease in the rotenone-insensitive pathway of approximately 65%. There was no significant change in the rate of rotenone-sensitive malate-FeCN reduction, which is catalyzed by Complex I of the respiratory chain. In addition, an immunoblot of mitochondrial proteins probed with antisera against the 51-kD subunit of bovine Complex I indicated that protein levels of this subunit were not different between wt and Atndi1 mutant mitochondria (data not shown). The activity of the external-facing inner membrane NADH DH was approximately 10-fold higher than external-facing inner membrane NADPH DH activity for both wt and Atndi1 mutant mitochondria (Fig. 5, A and B), but both of these activity ratios were approximately 4-fold greater in mutant mitochondria. No significant differences were observed in outer membrane NADH and NADPH DH activities between wt and mutant mitochondria (Fig. 5, C and D).
To confirm the location of the AtNDI1 protein, in vitro import assays were carried out. The full-length cDNA for AtNDI1 was cloned and sequenced using primers located in the 5'- and 3'-UTRs of the gene. Comparison of the cloned sequence with that from the database confirmed that the complete cDNA for AtNDI1 had been successfully and completely amplified (data not shown). In vitro translation of the cDNA produced a precursor protein with an apparent molecular mass of 60 kD (Fig. 6, lane 1). Upon incubation of the precursor with mitochondria, a product with an apparent molecular mass of 56 kD was produced, which was insensitive to digestion by added protein K (PK) (Fig. 6, lanes 2 and 3). The generation of this product was sensitive to valinomycin in the import assay, indicating that the protein was imported into or across the inner membrane (Fig. 6, lanes 4 and 5). A similar pattern was observed with the AOX precursor and for TIM23 except that the latter has no cleavable presequence; thus, no change in apparent mass was observed upon import.
To determine if the imported AtNDI1 was on the outside or inside of the inner membrane, we ruptured the outer membrane and prepared intermembrane space-depleted mitochondria to allow access of added PK to the outside of the inner membrane. Under these conditions, TIM23 was partially degraded indicated by a clear inner membrane-protected fragment, showing that the PK had gained access to intermembrane space components (Fig. 6, lane 3 verse 7). In contrast, under identical conditions, neither AOX nor AtDNI1 were digested by PK, indicating complete protection by the inner membrane. To further verify the intactness of our mitochondria and the rupture of the outer membrane, we carried out western-blot analysis with an outer membrane component (TOM20), an intermembrane space component (cytochrome c), and a matrix-located component (HSP70). It was observed that no significant rupture of the inner membrane had taken place under the conditions used because the HSP70 was intact in all the fractions. Likewise, with intact mitochondria, cytochrome c was protected from PK digestion. Most of this protein was lost from salt-washed intermembrane space-depleted mitochondria, and the small amount remaining was digested by PK with treatment of intermembrane space-depleted mitochondria. Finally, it was observed that TOM20 was accessible to PK in both mitochondria and intermembrane space-depleted mitochondria. The results together indicate that the PK treatment was sufficient to digest exposed proteins in the intermembrane space-depleted mitochondria. Thus, we conclude that the AtNDI1 was located on the inside of the inner membrane.
Identification of an Internal NAD(P)H DH In this study, evidence has been presented that directly links function with sequence information for the non-phosphorylating NAD(P)H DH from plant mitochondria. Three separate lines of evidence reported here suggest very strongly that the AtNDI1 gene encodes an NAD(P)H DH located on the inside of the mitochondrial inner membrane. First, the predicted protein sequence grouped with other suspected internal NAD(P)H DH such as ScNDI1 and potato NDA sequence. Second, the Atndi1 mutant containing an insert in the gene-encoding protein AtNDI1 has severely decreased rates of internal rotenone-insensitive NAD(P)H DH activity. Based on the molecular data presented (Fig. 3), the Atndi1 mutant contains an insert in the gene encoding the protein AtNDI1; as a consequence, this gene was not transcribed. On the other hand, transcription of At2g29990, which encodes a second, highly homologous, putative internal NAD(P)H DH, was unaffected. Thus, the loss of matrix NAD(P)H activity in the Atndi1 mutant could be attributed to the At1g07180 gene. Activity of both inner and outer membrane external NAD(P)H DH was either stimulated or unchanged in the mutant plants. Finally, the in vitro translation product of AtNDI1 was imported into isolated mitochondria and protected from proteinase treatment in mitochondria from which the outer membrane had been removed, confirming that this gene encodes a matrix-located protein. This enzyme consequently has been designated AtNDI1, in keeping with the nomenclature assigned for NADH DHs characterized in other species (particularly yeast). We propose that this nomenclature be applied to all non-phosphorylating NAD(P)H DHs identified in plants, once biochemical evidence has been provided for function and location.
There was not a complete elimination of the rotenone-insensitive pathway in the Atndi1 mutant (Fig. 4), but it is possible that the residual rate was generated from the gene product of the other NDA-like NAD(P)H DH, AT2g29990, suggesting that it can compensate for the lack of AtNDI1. This observation perhaps is not surprising given the highly similar sequences of the two putative internal NAD(P)H DH genes. The high level of homology between AT2g29990 and AtNDI1 is thought to be the consequence of a duplication event in the Arabidopsis genome. It is possible that subsequent to this event, the two genes have maintained function and evolved independently. However, the presence of two separate DHs on the inner matrix surface has only been demonstrated in potato and pea (Pisum sativum) leaf mitochondria (Melo et al., 1996
Measurement of the maximum capacity of matrix NAD(P)H oxidation is difficult and complex, and anumber of different strategies have been developed to determine this activity. In this project, we used a malate-FeCN reductase assay to determine matrix NAD(P)H oxidation and optimized malate DH and malic enzyme activities to ensure generation of sufficient NAD(P)H within the mitochondria to drive the low-affinity rotenone-insensitive enzymes (Møller and Palmer, 1982
The AtNDI1 sequence possesses two regions that meet the criteria for a dinucleotide-binding fold (DNF) domain, namely: (a) a Gly-rich consensus (GxGxxG), (b) a negatively charged residue (D or E) at the end of the second
It has been proposed that if one of the motifs is an NADPH-specific-binding site, then the third Gly in the Gly-rich consensus motif would generally be replaced by an S, A, or P and also that the negative charge at the end of the second
The interaction between the non-phosphorylating enzymes in plant mitochondria appears to be more complex than previously anticipated. Both the residual internal NAD(P)H oxidation rate and the increase in external NAD(P)H DH activities in the Atndi1 mutant illustrate this. Rotenone-sensitive Complex I activity was not affected in mitochondria from the mutant, yet overall matrix NAD(P)H oxidation decreased. This suggests that the rotenone-insensitive bypass contributes to electron flow in the absence of the inhibitor (Bryce et al., 1990
Sequence Analysis
BLAST (Altschul et al., 1990
Computer programs used to predict cellular location of proteins were: TargetP (Emanuelsson et al., 2000
Arabidopsis ecotype Columbia (wt) was used for all experiments. BLAST analysis of the Syngenta (San Diego) T-DNA insertion mutation database indicated that seed line SAIL_779_G05 contained an insert in either At1g07180 and/or At2g29990 (Sessions et al., 2002
Arabidopsis seeds were surface sterilized in 70% (v/v) ethanol for 2 min, soaked in 15% (v/v) bleach for 15 min, and then rinsed four to five times in sterile water. Shaking culture growth medium (40 mL; one-half-strength Murashige and Skoog basal salt mixture salts [Sigma, Australia], B5 vitamins, 2% [w/v] Suc, and 2 mM MES [pH 5.8]) was inoculated with approximately 50 sterile seeds. Cultures were grown for 14 d, with 18 h of light/6 h of dark at 23°C, with shaking at 80 rpm.
RNeasy and DNeasy kits (Qiagen USA, Valencia, CA) were used according to the manufacturer's protocols. DNA and RNA were run on a 1% (w/v) agarose gel to check quality and quantified spectrophotometrically.
Amplification reactions were carried out using primers described in Table II. Total RNA (3 µg) was used to synthesize cDNA using Invitrogen Superscript II Reverse Transcriptase and Oligo(dT)12-18 according to the manufacturer's protocols. PCR was carried out in the presence of 1.5 mM MgCl2, 0.2 mM of each dNTP, 100 ng of forward primer and reverse primer, 0.5 units of Taq DNA polymerase (Qiagen USA), and 20 ng of cDNA, in a final volume of 20 µL. Amplifications were performed on a Cetus 2400 thermal cycler (Perkin-Elmer Applied Biosystems, Foster City, CA) with the following cycling parameters: prestep at 95°C for 5 min; 20 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s; and an extension step of 72°C for 10 min. Amplification products (5-µL aliquots) were separated on a 1.5% (w/v) agarose gel. DNA size markers were run alongside the amplification products to determine fragment size.
The full-length cDNA (including start and stop codons) was amplified using the AT6-5'-UTR-F and AT6-3'-UTR-R primers with Arabidopsis leaf RNA as the starting template (RT-PCR carried out as described above). The amplification product was cloned into pGEMT-Easy (Promega, Madison, WI) for sequencing and pGEM3Zf(-) for translation and import experiments, using standard protocols (Sambrook et al., 1989
Mitochondria from 7-d-old soybean (Glycine max) cotyledons were isolated according to Day et al. (1985
Salt-washed intermembrane space-depleted mitochondria were prepared based on a procedure that removes most intermembrane space components in potato (Solanum tuberosum; Lister et al., 2002
Mitochondria were extracted from approximately 20 to 30 g of cultured Arabidopsis seedlings (root and shoot), washed, and purified on discontinuous Percoll gradients essentially as described by Soole et al. (1990
All assays were carried out spectrophotometrically using isolated, intact mitochondria in a standard reaction medium (0.25 M Suc, 0.01 M KH2PO4, 0.1 M TES, and 5 mM MgCl2 [pH 7.2]), unless otherwise stated. Succinate-DCPIP reductase was measured at 600 nm. Mitochondria were incubated on ice for 10 min with 20 mM succinate and 1 mM ATP. The rate was determined after the addition of 70 µM DCPIP, 0.3% (w/v) phenazine methosulphate, and azide. Rates were calculated using an extinction coefficient of 19.1 mM-1 cm-1. Malate-FeCN reductase was measured at 420 nm. Reactions were carried out at pH 6.8 in the presence of 1 mM NAD+, 10 mM malate, 10 mM pyruvate, 10 mM acetyl-CoA, thiamine pyrophosphate [to generate internal NAD(P)H via malate DH and malic enzyme], 1 mM KCN, 10 µM octyl gallate, and 0.9 mM FeCN. Rates were measured in the presence and absence of 25 µM rotenone. Butylmalonate (10 mM) was added at the end of the assay to inhibit malate transport across the membrane. Any residual rate was subtracted from the rate in the absence of the inhibitor to ensure that the NAD(P)H: FeCN reductase rate measured was not being generated by outer membrane or external-facing inner membrane NAD(P)H DHs. Rates were calculated using an extinction coefficient of 1.05 mM-1 cm-1. Rotenone-sensitive malate-FeCN reductase activity was determined by subtracting the rate in the presence of rotenone from the rate in the absence of rotenone. NAD(P)H-O2 was measured following NAD(P)H oxidation at 340 nm after the addition of 0.1 mM NADH or NADPH, 0.1 mM ADP, and 1 mM CaCl2. Rates were calculated using an extinction coefficient of 6.22 mM-1 cm-1 (for NADH and NADPH). NAD(P)H-cytochrome c reductase of the outer membrane was measured in the presence of 70 µM cytochrome c, 6 µM myxothiazol, and 1 mM KCN, following the addition of 1 mM NADH or NADPH, by monitoring the reduction of cytochrome c at 550 nm. Rates were calculated using an extinction coefficient of 19.8 mM-1 cm-1. All mitochondria preparations and assays were performed in triplicate. To normalize the data between mitochondrial preparations, the results for all assays are expressed as a ratio of the state 3 (+ADP) succinate:DCPIP reductase rate for the same mitochondrial preparation. The rates of succinate:DCPIP reductase activities were not significantly different between the wt and mutant mitochondrial preparations (P < 0.05, Mann Whitney t test).
The authors wish to acknowledge Syngenta (San Diego) for providing the T-DNA insert mutant seed. We would particularly like to thank David Oliver for his advice, especially with the seedling shaking cultures, and helpful feedback on the manuscript. Received June 30, 2003; returned for revision July 18, 2003; accepted August 17, 2003.
1 This work was fully funded by the Australian Research Council. Experiments were performed at Flinders University, with the exception of the in vitro translation and protein import studies, which were conducted at the University of Western Australia, Perth.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.029363. * Corresponding author; e-mail kathleen.soole{at}flinders.edu.au; fax 61-0-8-8201-3015.
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-sheet, and (c) six positions typically occupied by small hydrophobic residues (Bellamacina, 1996
