Two Cys or Not Two Cys? That Is the Question; Alternative Oxidase in the Thermogenic Plant Sacred Lotus

Sacred lotus ( Nelumbo nucifera ) regulates temperature in its ﬂoral chamber to 32 (cid:1) C to 35 (cid:1) C across ambient temperatures of 8 (cid:1) C to 40 (cid:1) C with heating achieved through high alternative pathway ﬂuxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys 1 and Cys 2 , are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys 1 with a -keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys 1 . Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b . Deduced amino acid sequences of both conﬁrmed that Cys 1 had been replaced by serine; however, Cys 2 was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys 1 and Cys 2 . An additional cysteine was present at position 193 in NnAOX1b. The signiﬁcance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.

Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32°C to 35°C across ambient temperatures of 8°C to 40°C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys 1 and Cys 2 , are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys 1 with a-keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys 1 . Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b. Deduced amino acid sequences of both confirmed that Cys 1 had been replaced by serine; however, Cys 2 was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys 1 and Cys 2 . An additional cysteine was present at position 193 in NnAOX1b. The significance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.

Thermogenesis in Sacred Lotus
Sacred lotus (Nelumbo nucifera) is a thermogenic plant that regulates the temperature of its floral chamber between 32°C and 35°C for up to 4 d (Seymour and Schultze-Motel, 1996). Heating of plant tissues has been described as an adaptation to attract insect pollinators either by volatilization of scent compounds (Meeuse, 1975) or by providing a heat reward (Seymour et al., 1983), protect floral parts from low temperatures (Knutson, 1974), or provide the optimum temperature for floral development (Ervik and Barfod, 1999;Seymour et al., 2009). In sacred lotus, heat is produced by high rates of alternative pathway respi-ration (Watling et al., 2006;Grant et al., 2008); however, the mechanisms of heat regulation, which likely occur at a cellular level, remain unclear.

Alternative Oxidase
Alternative pathway respiration is catalyzed by the alternative oxidase protein (AOX), which acts as a terminal oxidase in the electron transport chain but, unlike the energy conserving cytochrome pathway (COX), complexes III and IV are bypassed and energy is released as heat. Traditionally, AOX activity was measured using oxygen consumption of tissue, cells, or isolated mitochondria in the presence or absence of AOX and COX inhibitors. However, this method does not accurately measure activity in vivo but does indicate the capacity of the alternative pathway Day et al., 1996). The only method to date to accurately determine AOX activity, that is, flux of electrons through the AOX pathway in vivo, is to use oxygen isotope discrimination techniques (for review, see Robinson et al., 1995). Determining AOX activity in vivo is important because heat production in plants could be due to activity of either the AOX and/or plant uncoupling proteins. Using oxygen fractionation techniques, we have shown that flux through the AOX pathway is responsible for heating in sacred lotus (Watling et al., 2006;Grant et al., 2008). Furthermore, we were unable to detect any uncoupling pro-tein in these tissues (Grant et al., 2008). AOX protein content within the sacred lotus receptacle increases markedly prior to thermogenesis, but it remains constant during heating (Grant et al., 2008), suggesting that regulation of heating occurs through posttranslational modification of the protein.

Posttranslational Regulation of AOX Protein
The plant AOX is a cyanide-insensitive dimeric protein located in the inner mitochondrial membrane (Day and Wiskich, 1995). The dimer subunits (monomers) can be linked via a noncovalent association (reduced protein) or covalently through the formation of a disulfide bridge (oxidized protein; Umbach and Siedow, 1993). The reduced protein when run on SDS-PAGE has a molecular mass of approximately 30 to 35 kD and the oxidized protein 60 to 71 kD; this holds true for AOX from a number of species, including soybean (Glycine max) roots and cotyledons (Umbach and Siedow, 1993), tobacco (Nicotiana tabacum) leaf (Day and Wiskich, 1995), and the thermogenic spadix of Arum maculatum (Hoefnagel and Wiskich, 1998).
Regulation of AOX has been well studied in nonthermogenic plant species, and two mechanisms have been identified. Most AOX isoforms have two highly conserved Cys residues, Cys 1 and Cys 2 (defined in Berthold et al., 2000 andHoltzapffel et al., 2003), located near the N-terminal hydrophilic domain of the protein. In these isoforms, Cys 1 can either be reduced on both subunits of the AOX dimer, or the Cys 1 sulfhydryl groups can be oxidized to form a disulfide bridge (Rhoads et al., 1998). Reduction/ oxidation modulation of AOX in vitro can be achieved using the sulfhydryl reductant dithiothreitol (DTT) to reduce the protein or diamide to oxidize the Cys residues. The reduced dimer can be further activated via the interaction of Cys 1 with a-keto acids, principally pyruvate (Rhoads et al., 1998;see McDonald [2008] for a model of posttranslational regulation of AOX). In addition, Cys 2 may also be involved in regulating AOX activity through interaction with the a-keto acid glyoxylate (which can also stimulate activity at Cys 1 ; Umbach et al., 2002).
Recently, however, AOX proteins with different regulatory properties have been reported. Naturally occurring AOX proteins without the two regulatory Cys residues have been identified and, along with sitedirected mutagenesis studies, used to further elucidate the specific roles of Cys 1 and Cys 2 . The LeAOX1b isoform from tomato (Lycopersicon esculentum), which has a Ser residue at the position of Cys 1 and thus does not form disulfide linked dimers, is also activated by succinate rather than pyruvate when expressed in Saccharomyces cerevisiae (Holtzapffel et al., 2003). In Arabidopsis (Arabidopsis thaliana), uncharged or hydrophobic amino acid substitutions of either Cys result in an inactive enzyme, while positively charged substitutions produce an enzyme with higher than wild type basal activity but that is insensitive to pyruvate or succinate (Umbach et al., 2002). Single substitutions at Cys 1 or Cys 2 have revealed that glyoxylate can activate AOX via both Cys residues, but only one is needed for glyoxylate stimulation (Umbach et al., 2002(Umbach et al., , 2006. Double substitution mutants were not stimulated by either pyruvate or glyoxylate (Umbach et al., 2006).
Previously, we determined that thermogenesis via the AOX pathway in the sacred lotus receptacle is precisely regulated through changes in AOX flux rather than changes to protein content (Grant et al., 2008). In this study, we investigated the nature of this regulation in mitochondria isolated from heating receptacles. Our aim was to elucidate the reduction/ oxidation behavior of the AOX protein and the mechanisms of activation of cyanide-resistant respiration in sacred lotus receptacles to provide insights into the mechanism(s) of heat regulation in this species. We further investigated AOX regulation by determining the amino acid sequence of two novel AOX genes isolated from thermogenic receptacle tissue of sacred lotus.

Activity of Sacred Lotus AOX Is Stimulated by Succinate But Not Pyruvate or Glyoxylate
Residual mitochondrial respiration rates were quite low (,10 nmol O 2 min 21 mg 21 protein). Addition of NADH and KCN stimulated activity to an average of 50 nmol O 2 min 21 mg 21 protein, but this stimulation was not statistically significant. No stimulation was observed with subsequent addition of 5 mM pyruvate ( Fig. 1) nor with concentrations of pyruvate up to 20 mM. Addition of succinate, however, produced a 4-fold increase in activity to a mean of 196 6 20 nmol O 2 min 21 mg 21 protein (F 2,20 = 48.70, P , 0.0001; Fig. 1). Activation of respiration by succinate was similar in the presence or absence of malonate, which was used to inhibit complex II. Cyanide-resistant O 2 uptake was not stimulated by the addition of glyoxylate, either before or after succinate stimulation ( Fig. 1, C-F), and there was no increase in mitochondrial O 2 uptake with the possible substrates: citrate, fumarate, oxalate, a-ketoglutarate, or malate (data not shown). Manipulation of AOX redox state by addition of the sulfhydryl redox reagents DTT (Fig. 1B) and diamide had no effect on O 2 uptake. Cyanide-resistant oxygen uptake was almost completely inhibited by the AOX inhibitor n-propyl gallate ( Fig. 1, B, D, and F). however, most of the protein was insensitive to diamide even at high concentrations (50-250 mM). In contrast, treatment with the Lys-Lys-specific crosslinker ethylene glycol bis(succinimidylsuccinate) (EGS; 1 mM) caused 76% dimerization of the AOX protein (Fig. 2, lane 4).

Two Novel AOX Isoforms Lacking Cys 1 Occur in Thermogenic Sacred Lotus Tissue
Reverse transcription-PCR-based cloning of AOX transcripts was performed with total RNAs from thermogenic receptacles. Because two highly homologous partial fragments were detected during PCR analyses, full-length cDNAs of the corresponding transcripts were isolated and consequently named NnAOX1a and NnAOX1b (DNA data bank of Japan accession nos. AB491175 and AB491176, respectively). The deduced amino acid sequences of the encoded proteins indicate that NnAOX1a and NnAOX1b encode proteins of 39.0 and 39.3 kD, respectively, and 32.5 and 32.6 kD after cleavage of the mitochondrial targeting sequence. Both NnAOX1a and NnAOX1b contain some of the structural features typical of plant AOXs, such as four a-helical bundles and ligands for the two iron atoms of the active center (Moore and Albury, 2008). However, both NnAOX1a and NnAOX1b were found to contain a Ser residue at the site of the highly conserved Cys 1 residue, which is necessary for the regulation of the plant AOX through both redox control and a-keto acid stimulation, although the second conserved Cys, Cys 2 , was present in both (Fig. 3). Additionally, in the case of NnAOX1b, a Leu residue at position 193 was substituted by Cys (Fig. 3).

NnAOX1a and NnAOX1b Are Similar to AOX Isoforms from Other Dicots
Sequence alignment indicated that NnAOX1a and NnAOX1b are distinct from AOX isoforms reported from other thermogenic species in that they lack Cys 1 , while Dracunculus vulgaris, Philodendron bipinnatifidum, Sauromatum guttatum, and Symplocarpus reinifolius all contain both Cys 1 and Cys 2 (Fig. 3). Further analysis indicated that NnAOX1a and NnAOX1b from thermogenic sacred lotus were more similar to AOXs from other dicots than they were to AOXs from other thermogenic plants ( Fig. 4; Supplemental Fig. S1). Figure 1. Rate of O 2 uptake in mitochondria from thermogenic sacred lotus receptacles. Each column (means 6 SE, n = 3-4) represents a subsequent addition to the O 2 electrode chamber of 2 mM NADH, 5 mM pyruvate (Pyr), or 20 mM succinate (Succ) and/or 5 mM glyoxylate (Glyox). These additions were made in the presence of 1 mM KCN. Columns with different letters are significantly different. Typical O 2 uptake traces per milligram of mitochondrial protein are shown to the right of the graphs. Numbers below the traces are the respiration rates (nmoles O 2 min 21 mg protein 21 ). nPG, n-Propyl gallate. NnAOX1b also contains an extra Cys residue at position 193; this is similar to AtAOX1a, LeAOX1a, LeAOX1b, and NtAOX1a, in which a Leu is replaced by Cys at the same position (Supplemental Fig. S1). Based on the AOX model for S. guttatum (Andersson and Nordlund, 1999), this Cys is located after the first a-helix, but we are unsure whether it sits within the membrane or matrix region of the protein.

DISCUSSION
In most plants studied to date, the a-keto acid pyruvate stimulates AOX activity (Day et al., 1994), and the specific site of this regulation is reduced Cys 1 (Rhoads et al., 1998). Following pyruvate stimulation, glyoxylate can further increase AOX activity via Cys 2 (Umbach et al., 2002) and can also initiate activity at either Cys alone (Umbach et al., 2006). Recent studies on AOX isoforms without the regulatory Cys residues have revealed stimulation by succinate, not pyruvate, when Cys 1 is not present (Djajanegara et al., 1999;Holtzapffel et al., 2003), and the glyoxylate effect is absent when both Cys residues are missing (Umbach et al., 2006). Here, we report that AOX from thermogenic tissues of sacred lotus is stimulated by succinate rather than pyruvate (Fig. 1A), that there is no glyoxylate effect (Fig. 1, C-F), and that the majority of AOX could not be reversibly reduced and oxidized (Fig. 2). Our results thus suggested that the majority of AOX in these tissues lacked Cys 1 and that Cys 2 might also be missing. Subsequent sequencing of two cDNAs, NnAOX1a and NnAOX1b, isolated from thermogenic sacred lotus indicated that Cys 1 is replaced by Ser but that Cys 2 is present in both (Fig. 3). This confirmed our predictions, based on the in vitro studies of isolated mitochondria, that Cys 1 was missing from the majority of AOX protein in these tissues. The situation with Cys 2 is complicated, however, by the fact that glyoxylate stimulation of AOX containing this residue varies between naturally occurring and site-directed AOX substitutions. For example, similarly to our experiments, glyoxylate failed to stimulate tomato AOX (LeAOX1b) even though it contains Cys 2 (Holtzapffel et al., 2003). LeAOX1b was also activated by succinate in a similar fashion to the thermogenic lotus AOX. In contrast, site-directed mutation of both Cys residues in Arabidopsis indicated that only one Cys was needed for glyoxylate stimulation (Umbach et al., 2002(Umbach et al., , 2006. In the majority of plants, AOX can be reversibly reduced and oxidized (Umbach and Siedow, 1997). However, when extracted under nonreducing conditions, the sacred lotus receptacle AOX protein was predominantly in the reduced (i.e. nonlinked) state and could not be further oxidized with diamide across a range of concentrations up to 250 mM. This contrasts strongly with AOX proteins from soybean cotyledons and Arabidopsis leaves, where diamide concentrations of ,5 mM were sufficient to oxidize AOX (Umbach and Siedow, 1993), while 200 mM diamide was able to oxidize AOX protein from chilled green tomato mitochondria (Holtzapffel et al., 2003). As formation of the oxidized dimer requires the presence of Cys 1 (Rhoads et al., 1998;Djajanegara et al., 1999;Umbach et al., 2006), our results are consistent with this regulatory Cys being absent from the majority of AOX found in thermogenic sacred lotus. This results in an AOX that is permanently in the reduced state and ready for further activation by succinate. Thus, fine control of activity during heating may be modulated by succinate levels. Similarly, naturally occurring and mutated AOX proteins with Ser substitutions at Cys 1 lack the ability to form oxidized dimers and, like the sacred lotus receptacle AOX, are poised for activation (Ito et al., 1997;Umbach et al., 2002;Holtzapffel et al., 2003;Umbach et al., 2006).
In contrast to the results with diamide, AOX from the sacred lotus receptacle was able to form dimers when exposed to the Lys-Lys cross-linker EGS. Monomeric AOX proteins such as those found in fungi (e.g. Neurospora crassa and Pichia stipitis) do not form dimers in the presence of EGS or diamide (Umbach and Siedow, 2000). Thus, while most of the thermogenic sacred lotus AOX protein is able to be covalently bound, only a small fraction (approximately 20%) can form disulfide bonds in the presence of diamide (Fig.  2). This suggests that there may be an additional isoform that unlike NnAOX1a and NnAOX1b contains Cys 1 . Alternatively, there is the possibility that the additional Cys at position 193 in NnAOX1b may be involved in disulfide bridge formation, although this Cys may not be close enough to Cys 2 in the tertiary or quaternary structure of the protein to form disulfide bonds (Gilbert, 1990). Interestingly, a further isoform may be present in thermogenic sacred lotus, as we detected a small band around 60 kD that could not be reduced in the presence of DTT (Fig. 2, lane 2). This band represented around 12% of the total AOX protein present in our samples. Multiple AOX isoforms in the same tissue have been reported in a number of different species, including thermogenic S. guttatum, in which a 37-kD species is joined by a 35-and 36-kD species during thermogenesis (Rhoads and McIntosh, 1992). It is possible that these different isoforms could form heterodimers. A mixture of homodimers and Figure 3. Deduced amino acid sequences of NnAOX1a and NnAOX1b aligned with those of previously reported AOXs expressed in thermogenic tissues. Bold characters highlight residues conserved across all of the AOX sequences in the alignment. The putative structural features are indicated as follows: asterisks for two highly conserved Cys residues, termed Cys 1 and Cys 2 (Berthold et al., 2000), double underline for ligands to iron atoms of the catalytic center, and gray bars for four a-helices. Abbreviations and data sources are as follows: DvAOX, D. vulgaris AOX (BAD51465); PbAOX, P. bipinnatifidum AOX (BAD51467); SgAOX, S. guttatum AOX (P22185); SrAOX, S. renifolius AOX (BAD83866). heterodimers have been proposed to occur in soybean (Finnegan et al., 1997), while in tomato it was suggested that heterodimeric associations between LeAOX1a and LeAOX1b could explain why full oxidation of tomato AOX dimers did not occur (Holtzapffel et al., 2003). Whether AOX heterodimers occur in thermogenic sacred lotus and whether they have different catalytic properties from homodimers has yet to be investigated. Crichton et al. (2005) suggested that changes to amino acids other than the regulatory Cys 1 and Cys 2 may influence AOX activity in thermogenic species. This suggestion is based on a constitutively active SgAOX, with both conserved Cys residues, which when expressed in yeast was insensitive to both pyruvate and succinate. However, the absence of Cys 1 in both NnAOX1a and NnAOX1b, and the fact that succinate was required for full alternative pathway activity in mitochondria isolated from thermogenic sacred lotus, make it unlikely that these isoforms are regulated in a similar way to that hypothesized for S. guttatum (Crichton et al., 2005). Furthermore, AOX proteins that have been modified by amino acid substitutions or expressed in bacteria or yeasts may not reflect in vivo behavior; thus, comparisons with naturally occurring isoforms need to be approached with caution. Unlike previous studies, the AOX lacking Cys 1 in sacred lotus is a naturally occurring isoform expressed in plant mitochondria.
Our observation that succinate stimulation of AOX occurs in thermogenic sacred lotus mitochondria even in the presence of malonate (a succinate dehydrogenase inhibitor) suggests a possible nonmetabolic interaction of succinate with the AOX protein. As succinate is a common TCA cycle intermediate, it is possible that upstream substrate availability could be a signal for AOX activation. Other thermogenic species that are poised in the reduced state and that use lipids instead of carbohydrates to fuel thermogenesis, for example P. bipinnatifidum (N. Grant and R. Miller, unpublished data), may use products from lipid metabolism to signal AOX activation. If substrate supply is the signal, succinate activation of sacred lotus AOX may play a larger role than previously thought; however, this requires further investigation. Ubiquinol reduction status (Wagner et al., 2008) as well as regions in the AOX sequence located near the C terminus of the protein unique to thermogenic species (Crichton et al., 2005;Onda et al., 2008) could also be involved in controlled thermogenesis in these species.

CONCLUSION
Through a combination of biochemical and molecular techniques, we have investigated the regulation of AOX activity in thermogenic tissues of sacred lotus. This has enabled us to expand our understanding of how heating may be regulated in this and other thermoregulating species. The major isoforms of AOX found in lotus, NnAOX1a and NnAOX1b, lack Cys 1 and could therefore not form disulfide linked dimers. The lack of Cys 1 also explains the pyruvate insensitivity of alternative pathway respiration in thermogenic lotus and also suggests that present in NnAOX1b, does not substitute for pyruvate activation via Cys 1 . Our sequence data indicated that AOXs from thermogenic plants do not form a functional grouping and that heating in these plants may thus be a function of the amount of AOX protein present rather than the structure of the protein. Fine control of AOX activity in thermoregulating species is yet to be elucidated but may involve modulation by the organic acids pyruvate or succinate, depending on which isoform of the protein is present.

Plant Material
Lotus flowers (Nelumbo nucifera) were collected from an outdoor pond in the Adelaide Botanic Gardens, South Australia, in January and February 2007 to 2009. Flowers for mitochondrial measurements were collected early during the thermoregulatory period classified as stage 1 by Grant et al. (2008). Stage 2 flowers were used for isolation of total RNA.

Isolation of Mitochondria
Washed mitochondria were isolated from approximately 50 g of fresh sacred lotus receptacle tissue according to Day et al. (1985) with minor modifications (Grant et al., 2008). The mitochondria were purified using a three-step Percoll gradient (30 mL) made of equal amounts of 50% (v/v), 35% (v/v), and 20% (v/v) Percoll in a Suc wash buffer (250 mM Suc, 10 mM HEPES-KOH, pH 7.2, and 0.2% [w/v] fatty acid-free bovine serum albumin [BSA]). The gradients were centrifuged at 20,000g for 1 h at 4°C, and purified mitochondria were collected from the 20% to 35% interface. Mitochondria were then washed (0.4 M mannitol, 10 mM MOPS/KOH, pH 7.2, and 0.1% [w/v] fatty acid-free BSA) twice by centrifugation at 10,000g and the final pellet resuspended in 1 mL wash buffer. Mitochondrial protein was determined according to the method of Bradford (1976).

Treatment of Mitochondria with Diamide and DTT
Percoll purified mitochondria were left untreated or treated with either DTT, diamide, or EGS to final concentrations of 20, 10, and 5 mM, respectively. Higher concentrations of EGS completely cross-linked the AOX protein; however, the AOX signal was greatly reduced. A high dimethyl sulfoxide (DMSO)/protein ratio may have had a detrimental effect on the protein; therefore, lower concentrations of EGS were used. Following the addition of DTT, mitochondria were incubated on ice for 30 min. Mitochondria treated with EGS or diamide (30 min at room temperature) were incubated with DTT first, to ensure the AOX protein was in the reduced form, and then washed before addition of the aforementioned reagents. Reactions were quenched by adding excess Tris-HCl (1 M, pH 7.4). Stock solutions of diamide and EGS were prepared in DMSO. The DTT was prepared in purified water; however, DMSO was added to both DTT-treated and untreated mitochondria at the same final concentration as in the diamide treatment as a control. All solutions were prepared fresh on the day of use.

SDS-PAGE and Immunoblotting
Mitochondrial protein samples were separated by nonreducing SDS-PAGE gels and immunoblotted as previously described (Grant et al., 2008 bodies raised against Sauromatum guttatum AOX (Elthon et al., 1989) were used to detect the AOX protein. The proteins were visualized using Super-Signal west femto maximum sensitivity substrate (Pierce). All buffers were reductant free.

Mitochondrial Respiration Measurements
Oxygen uptake by purified mitochondria was measured at 25°C using a Clark-type oxygen electrode in 1.8 mL of reaction medium (0.2 M Suc, 10 mM KCl, 1 mM MgCl 2 , 5 mM KH 2 PO 4 , 20 mM MOPS/KOH, pH 7.2, and 0.1% [w/v] fatty acid-free BSA). The O 2 concentration in air-saturated buffer at 25°C was estimated at 250 mM in each experiment. Mitochondrial O 2 uptake was initiated with 2 mM NADH and 20 mM succinate (final cuvette concentration). Approximately 100 mg of mitochondrial protein was used in each assay. KCN at a final concentration of 1 mM was used to inhibit the COX pathway, and 100 mM n-propyl gallate was used to inhibit the AOX pathway. A steady state of O 2 uptake was reached before addition of subsequent constituents. Depending on the experiment, the following were added to the reaction mix (shown as final cuvette concentration): 20 mM pyruvate, 5 mM glyoxylate, 10 mM citrate, 10 mM fumarate, 10 mM oxalate, 10 mM a-ketoglutarate, 10 mM malate, 5 mM DTT, and 5 mM diamide. To account for the effect of residual pyruvate, lactate dehydrogenase (5 units/mL) was added to the reaction medium to scavenge residual pyruvate. Malonate (1-10 mM) was used to determine whether succinate was acting as a substrate for succinate dehydrogenase (complex II) or an activator of AOX. Initial experiments showed no evidence of state 3 to state 4 transition following the addition of ADP, and the succinate-stimulated O 2 uptake was not inhibited by KCN, suggesting that the bulk of respiration was occurring via the AOX pathway.
To obtain full-length cDNAs of NnAOX1a and NnAOX1b, PCR amplification was performed using KOD-Plus (TOYOBO). The final PCR products were subcloned into the HincII site of pUC118 (TaKaRa Bio) and their sequences determined. Nucleotide sequence data were analyzed with GENETYX software (Genetyx). Phylogenetic analyses of AOX sequence data were conducted using MEGA4 (Tamura et al., 2007). The phylogeny was deduced using the neighbor-joining method for 29 molecular species of AOX proteins and tested by bootstrap analysis with 500 replicates. Branches corresponding to partitions reproduced in ,50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All positions containing gaps and missing data were eliminated from the data set.

Statistical Analysis
Changes in mitochondrial activity with respect to different substrates were compared using one-way ANOVA (JMP 5.1; SAS Institute). Tukey's honestly significant difference post hoc tests were used to identify significantly differ-ent means. Data sets were tested for normality and homogeneity of variances using Shapiro-Wilk W and Bartlett's tests, respectively. Significant differences between means were calculated at P = 0.05.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AB491175 (NnAOX1a) and AB491176 (NnAOX1b).

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Sequence alignment of NnAOX1a, NnAOX1b, and AOX1 proteins from other dicot species.

ACKNOWLEDGMENTS
We thank the Adelaide Botanical Gardens for access to their lotus pond, Laura Howie for technical assistance, Associate Professor Kathleen Soole, and Professor James Whelan for the kind donation of AOX antibodies. We also thank two anonymous reviewers for their constructive and insightful comments.