The mitochondrial oxidative phosphorylation system in plants possesses a variety of alternative pathways that decrease respiratory ATP production. These alternative pathways are mediated by three classes of bypass proteins: the type II NAD(P)H dehydrogenases (which circumvent complex I of the electron transport chain), the alternative oxidases (AOXs; which circumvent complexes III and IV) and the uncoupling proteins (which circumvent ATP synthase). We have monitored the expression of all genes encoding respiratory bypass proteins in Arabidopsis thaliana growing with different sources of inorganic nitrogen (N). Resupply of nitrate (NO) to N-limited seedling cultures caused a decrease in the transcript abundance of several type II NAD(P)H dehydrogenase and AOX genes, while resupply of ammonium (NH) led to broad increases in expression in the same gene families. Similar results were observed upon switching between nitrate and ammonium in the absence of N stress. Nitrate signalling was found to be mediated primarily by the nitrate ion itself, whereas ammonium regulation was dependent upon assimilation and affected by changes in apoplastic pH. Corresponding alterations in alternative respiratory pathway capacities were apparent in seedlings supplied with either nitrate or ammonium as an N source and in mitochondria purified from the seedlings. Specifically, AOX capacity and protein abundance, as well as calcium-dependent external NADH oxidation, were substantially elevated after growth on ammonium. The increased capacity of respiratory bypass pathways after switching from nitrate to ammonium was correlated to an overall respiratory increase.
Plants require large amounts of nitrogen (N) for the biosynthesis of amino acids and secondary metabolites, and N availability, as nitrate (NO) or ammonium (NH), varies between ecosystems and limits plant growth. Most plants can take up and utilize either nitrate or ammonium, with the former transported to leaves for reduction and assimilation and the latter generally assimilated directly in roots (Crawford and Forde, 2002). However, nitrate has a higher energetic requirement for assimilation than ammonium, as nitrate must be reduced to nitrite in the cytosol (using one NADH) and then to ammonium in the plastid (using three NADPH equivalents). Ammonium, either generated from nitrate or taken up from the soil, is eventually incorporated into the amino acid glutamate by the glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) system. Considering that N assimilation rates are 5–13% of CO2 assimilation rates, nitrate reduction can constitute a major sink for cellular reductant (Noctor and Foyer, 1998).
Both substantial reductant demands and the GS/GOGAT requirement for 2-oxoglutarate (2-OG) as a carbon skeleton ties the process of N assimilation to cellular respiration. Indeed, the addition of ammonium or nitrate to whole plants or plant cells induces (at the transcript and/or activity level) enzymes in glycolysis and the Krebs cycle (e.g. pyruvate kinase, PEP carboxylase, citrate synthase and aconitase) that are required for the synthesis of 2-OG (Lancien et al., 1999; Larsen et al., 1981; Paul et al., 1978; Scheible et al., 1997). In concert, respiratory chain activity is enhanced in vivo (O2 consumption increases) upon addition of ammonium or nitrate to N-limited plants (Barneix et al., 1984; Bloom et al., 1992; Rigano et al., 1996; Scheible et al., 2004). Overall, these studies suggest that the manipulation of N nutrition can provide an excellent system to study dynamic alterations in plant respiratory metabolism in response to changes in cellular energetic demands.
Plant mitochondria contain several alternative pathways, all of which can be considered bypasses of one or more of the multiprotein complexes of oxidative phosphorylation: type II NAD(P)H dehydrogenases circumvent respiratory complex I, alternative oxidases (AOXs) circumvent complexes III and IV, and uncoupling proteins circumvent ATP synthase (also called complex V; Rasmusson et al., 2004; Vanlerberghe and McIntosh, 1997; Vercesi, 2001). Redox reactions catalysed by complexes I, III and IV are coupled to proton pumping across the inner mitochondrial membrane, and the resultant electrochemical gradient is harnessed by complex V for ATP production. In contrast, type II NAD(P)H dehydrogenases and AOXs do not pump protons, and proton flow through uncoupling proteins is not coupled to ATP synthesis. Therefore, flux through the alternative pathways does not contribute to respiratory ATP production and is not controlled by cellular adenylate status. Respiratory bypass proteins have been implicated in several physiological processes, including thermogenesis (Siedow and Day, 2000), the prevention of reactive oxygen species formation (Fernie et al., 2004; Møller, 2001) and the dissipation of excess redox equivalents (Raghavendra and Padmasree, 2003). Indirect evidence also suggests that rapid adjustments in the respiratory chain occur primarily through regulation of the alternative pathways: the multiprotein basal respiratory complexes appear to be relatively insensitive to external factors, while the respiratory bypasses display rapid quantitative changes in response to various biotic and abiotic stimuli (e.g. Escobar et al., 2004; Finnegan et al., 1997; Simons et al., 1999; Svensson and Rasmusson, 2001; Yu et al., 2001).
To obtain a comprehensive picture of how the N status may affect respiratory bypass pathways, we have used a sensitive real-time reverse transcript (RT)-PCR approach to monitor the transcript abundance of all members of the type II NAD(P)H dehydrogenase, uncoupling protein and AOX gene families. We found that nitrate and ammonium have strong and opposing effects on several respiratory bypasses that are manifested at the mRNA, protein, and activity levels.
A screen to identify nitrogen-regulated respiratory bypass genes
The growth of Arabidopsis seedlings in shaking liquid cultures allows the rapid manipulation of N availability and form in a sterile environment that precludes microbial nitrification reactions (the conversion of ammonium to nitrate). Thus, this system is ideal for analyses of rapid N-mediated effects on gene expression (Scheible et al., 2004; Wang et al., 2000). We transferred 9-day-old seedlings grown in complete nutrient medium to an N-free medium for 3 days and then added 9 mm KNO3, 4.5 mm (NH4)2SO4, or 4.5 mm K2SO4 (negative control) to the cultures. The relative transcript abundance of all type II NAD(P)H dehydrogenase, uncoupling protein and AOX genes was analysed by real-time RT-PCR 8 h after N (or K2SO4) resupply. The constitutively expressed gene encoding the 76-kDa subunit of respiratory complex I (Svensson and Rasmusson, 2001) was utilized as a negative control and Nia2, the nitrate-induced gene for nitrate reductase 2 (Crawford et al., 1988), was used as a positive control.
As summarized in Table 1, the addition of nitrate to the N-limited seedlings led to substantial (>twofold) decreases in the transcript abundance of the AOX gene aox1a and the type II NAD(P)H dehydrogenase genes nda2 and ndb4 as compared with the K2SO4 control. In contrast, resupply of N as ammonium caused twofold–fourfold increases in expression of aox1a, aox1d and ndb2, and a massive 15-fold increase in expression of aox2. Thus, with the exception of the ucp1 gene, which was slightly induced by both forms of N, there was a general trend of upregulation of respiratory bypass gene expression by ammonium but suppression of gene expression by nitrate. The aox1a, aox1d, aox2, nda2, ndb2 and ndb4 genes, whose transcript abundance varied >2.5-fold between the experimental treatments, were selected for further study.
Table 1. A real-time RT-PCR screen for nitrogen (N)–regulated respiratory genes
Relative transcript levels
Transcript levels were determined 8 h after supplementing N-limited seedlings with respective compounds. Transcript abundance was quantified in comparison to the K2SO4 control, which was arbitrarily assigned a value of 1. Data are given as mean ± SD (n = 2). For replicate treatment see Experimental procedures. A significant difference between (NH4)2SO4 and KNO3 transcript levels is denoted by an asterisk (P < 0.05). Ct, cycle threshold; ND, not consistently detected.
1.00 ± 0.05
0.58 ± 0.04
1.39 ± 0.33*
1.00 ± 0.10
0.36 ± 0.09
1.17 ± 0.17*
1.00 ± 0.02
1.07 ± 0.05
0.99 ± 0.05
1.00 ± 0.06
1.03 ± 0.14
2.69 ± 0.35*
1.00 ± 0.18
0.27 ± 0.01
1.50 ± 0.40*
1.00 ± 0.03
1.16 ± 0.18
1.01 ± 0.19
1.00 ± 0.01
0.46 ± 0.23
2.00 ± 0.14*
1.00 ± 0.12
1.67 ± 0.42
1.35 ± 0.27
1.00 ± 0.06
1.67 ± 0.29
1.28 ± 0.02
1.00 ± 0.11
0.65 ± 0.34
4.28 ± 1.19*
1.00 ± 0.02
1.38 ± 0.21
15.17 ± 1.88*
1.00 ± 0.01
2.09 ± 0.15
1.98 ± 0.07
1.00 ± 0.15
1.08 ± 0.07
0.96 ± 0.14
1.00 ± 0.14
1.55 ± 0.44
1.11 ± 0.02
1.00 ± 0.04
1.14 ± 0.13
0.98 ± 0.00
1.00 ± 0.05
3.14 ± 0.28
1.50 ± 0.35*
The kinetics and organ specificity of nitrogen effects on respiratory bypass genes
In order to verify initial screening results and to learn more about the kinetics of N regulation of the alternative respiratory pathways, the N starvation/resupply experiments described above were repeated and transcript abundance was monitored at 2, 8 and 24 h after addition of nitrate, ammonium, or potassium sulphate (Figure 1). Rapid responses to nitrate and/or ammonium, with clear alterations in transcript levels 2 h after N resupply, were obvious for all of the investigated respiratory bypass genes except for aox1d, which had a delayed response to ammonium. As expected, nda2 expression was suppressed by nitrate, aox2 and ndb2 expression was induced by ammonium, and expression of aox1a and aox1d was both induced by ammonium and suppressed by nitrate. Surprisingly, ndb4 was not downregulated by nitrate, as previously observed, but was markedly induced by ammonium (thus approaching an ammonium:nitrate transcript abundance ratio similar to that reported in Table 1).
To examine the organ specificity of N-mediated changes, RNA was isolated specifically from root and leaf tissue 8 h after N resupply. In general, transcript levels of nda2, aox1a and aox1d were higher in leaves, while the ndb2, ndb4 and aox2 transcripts were more abundant in roots (Figure 2, left column). The ammonium and nitrate effects described above are reflected very similarly in both roots and leaves, though with some differences in the magnitude of induction/suppression between the organs (Figure 2, right column).
Expression of respiratory bypass genes under non-nitrogen-limited conditions
The withdrawal of N from rapidly growing seedling cultures for a period of 3 days imparted a physiological stress, which was phenotypically manifested through typical N deficiency symptoms like leaf chlorosis, anthocyanin accumulation and root proliferation. In order to separate transcriptional responses related to N stress from specific effects arising from the uptake/assimilation of different forms of N, we maintained seedlings on a complete nutrient medium and alternated between nitrate and ammonium as the exclusive N source. As shown in Figure 3, transfer of N-sufficient seedlings from nitrate to ammonium nutrition led to rapid induction of aox1a, aox1d and ndb4, with the opposite effect occurring upon transfer from ammonium to nitrate nutrition. Expression of aox2 and nda2 rapidly decreased when plants were switched from ammonium to nitrate nutrition, but the reverse effect was not apparent 8 h after transfer from nitrate to ammonium. Only minor changes in transcript levels were observed for the type II NAD(P)H dehydrogenase gene ndb2, indicating that induction by ammonium (Figure 1) may require previous N deficiency/stress. Perhaps most important, when comparing seedlings continuously grown on nitrate (56 h) to seedlings continuously grown on ammonium (56 h), it is apparent that steady-state transcript levels of the respiratory bypass genes were maintained at very different levels depending upon the form of N nutrition (Figure 3, compare NO to NO with NH to NH).
Signalling pathways controlling opposing ammonium and nitrate responses
The assimilatory pathways for nitrate and ammonium obviously converge once nitrite is fully reduced to ammonium in the plastid. Thus, potential signalling intermediates downstream of the GS/GOGAT system, such as altered glutamine or glutamate levels (Coruzzi and Bush, 2001), cannot explain the contrary effects of ammonium and nitrate on gene expression. However, there are substantial physiological differences between nitrate and ammonium nutrition concerning both ion uptake and early assimilatory reactions (Crawford and Forde, 2002). The most significant of these differences are the higher energetic cost associated with nitrate assimilation (discussed above) and the opposing effects of nitrate and ammonium on soil pH. In order to maintain intracellular charge balance, ammonium uptake is accompanied by proton extrusion by a plasma membrane H+-ATPase, resulting in a rapid and substantial acidification of rhizosphere soil. In contrast, nitrate uptake occurs via proton cotransport or in exchange for OH− or HCO, resulting in rhizosphere soil alkalinization (Crawford and Forde, 2002; Hinsinger et al., 2003; Ruan et al., 2000). Corresponding pH effects were observed in our seedling cultures, as provision of ammonium to N-limited seedlings resulted in rapid acidification of the growth medium, while provision of nitrate resulted in medium alkalinization (Figure 4a). Similar, though less rapid, effects were observed in cultures in which the N source was alternated between ammonium and nitrate (Figure 4b).
In order to determine the role of uptake-associated changes in extracellular pH on respiratory bypass gene expression, we supplemented the seedling growth medium with 5 mm MES (pH 5.9), which almost completely buffered ammonium- and nitrate-mediated changes in pH. Nitrate resupply to N-limited, pH-buffered seedling cultures led to a decrease in expression of nda2, aox1a and aox1d, an effect essentially identical to what was observed in non-buffered medium (Figure 5a; compare to Figure 1). Thus, the nitrate-mediated downregulation of alternative respiratory pathways appears to be largely pH-independent. Surprisingly, nitrate increased the abundance of the ndb4 transcript in the absence of medium alkalinization, which contrasts with the lack of effect in non-buffered media (Figure 1). As for unbuffered cultures, the resupply of ammonium to MES-supplemented cultures caused increases in aox1a, aox1d, aox2, ndb2 and ndb4 transcript abundance. However, the quantitative magnitude of induction was either slightly (aox1a, aox1d, ndb2) or substantially (aox2) lower in the absence of medium acidification, suggesting that both extracellular pH and ammonium itself could play cooperative roles in signalling.
We attempted to further separate the effects of ammonium per se from the effects of medium pH by adding nitrate to N-starved seedling cultures and then maintaining medium pH at 6.3, 5.9, 5.5, 5.1, or 4.3 by regular addition of 1 m HCl (Figure 5b). Consistent with the interpretations above, the aox1d and aox2 genes were induced by a low extracellular pH (4.3) even in the absence of ammonium. The pH-mediated changes in aox1d and aox2 transcript levels were quite modest as compared with the joint effect of low pH and ammonium (Figure 1). Therefore, independent ammonium and pH signals both clearly contribute to the upregulation of these genes. Regulation of ndb4 also displayed significant pH sensitivity, with a steady increase in transcript abundance with rising medium acidity. Given the results summarized in Figure 5(a,b), the interaction between N and extracellular pH in the regulation of ndb4 expression appears quite complex and likely involves additional regulatory factors. Extracellular pH had no substantial effects on the aox1a, nda2 and ndb2 genes, suggesting that pH has no major signalling role for these genes, at least in the absence of ammonium.
Having established that nitrate-mediated changes in respiratory bypass gene expression are pH-independent, we utilized tungstate, a specific inhibitor of nitrate reductase, to define potential nitrate-derived signalling candidates (Glass et al., 2002; Vidmar et al., 2000). The addition of 0.5 mm tungstate (as Na2WO4) to N-limiting seedling cultures 2 h prior to nitrate resupply did not inhibit nitrate-mediated transcript reductions for aox1d and nda2, and only partially for aox1a (Figure 5c). These results suggest that at least aox1d and nda2 are directly responsive to the nitrate ion and not to a product of its assimilation. For the corresponding analysis of ammonium induction, we used the GS inhibitor methionine sulfoximine. This inhibitor completely abolished ammonium induction of aox1a, aox1d and ndb2 and substantially reduced the magnitude of aox2 and ndb4 induction (Figure 5d), suggesting that ammonium response involves elements downstream of the GS/GOGAT step. It further indicates that the pH-independent ammonium induction of alternative pathways is not caused by futile ATP-consuming proton cycling over the plasma membrane, because methionine sulfoximine has no effect on this futile cycling (Britto et al., 2001; Kronzucker et al., 2001).
Ammonium induction of the alternative oxidase pathway capacity in seedlings
From a physiological perspective, transcript-level analyses are of limited value without confirmation of corresponding changes in enzyme activities. The genes encoding three of the five AOX proteins in Arabidopsis are responsive to nitrate, ammonium, or both, so substantial alterations in AOX capacity would be expected in plants grown under different N nutrition regimes. Arabidopsis seedling cultures, grown in complete medium for 8 days, were transferred to a medium containing either 4.5 mm nitrate or 4.5 mm ammonium for 2 days (Figure 6). At this stage, the AOX capacity was 4.5 times higher (248 versus 53 nmol O2 consumed min−1 gFW−1) in ammonium-treated than in nitrate-treated seedlings, and total seedling respiration was consistently higher in the ammonium-treated seedlings (393 versus 285 nmol O2 consumed min−1 gFW−1; Figure 6, compare NHcontrol with time zero in the induction curve). Thereafter, nitrate-treated seedlings were transferred to ammonium medium and incubated for 8–48 h. During this ammonium treatment, the alternative pathway gradually increased, almost fourfold, ending at a level slightly below the capacity of the NH control described above. During the ammonium treatment, total seedling respiration also increased, consistent with the AOX capacity induction (Figure 6). However, the AOX induction was relatively slow and there was a lag phase before an increase became visible in total tissue respiration. We therefore analysed the persistence of nitrate in the seedlings after the transfer to ammonium (Figure 6). The results show that most residual nitrate is assimilated during the first day after the switch to ammonium medium and indicates that nitrate suppression of the expression of aox genes (Table 1) may delay the induction by ammonium. Overall, consistent with the transcript data, AOX capacity is dramatically increased in seedlings grown under ammonium nutrition, leading to an increase in total respiration.
Changes in protein abundance and respiratory activities in purified mitochondria
In order to further assess N-mediated changes in respiratory activity, mitochondria were purified from seedlings treated with nitrate or ammonium for 2 days, as for the O2 consumption measurements. Using a modified protocol for mitochondrial isolation, it was possible to purify coupled mitochondria with functionally intact inner membranes. This is seen from the stimulation of external NADH oxidation by ADP (allowing the ATP synthase to decrease the proton gradient over the inner membrane), the subsequent rate decrease by the ATP synthase inhibitor oligomycin, and the final 2.2–2.5-fold stimulation by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler that abolishes the proton gradient (Figure 7).
Western analysis demonstrated that mitochondria from ammonium-treated seedlings contain much higher levels of AOX protein than mitochondria from nitrate-treated seedlings (Figure 8). The signals for the NAD9 subunit of complex I and subunit 1 of complex IV (COX1) were similar between the two treatments, suggesting that these basal respiratory complexes were unaffected by the N source, at least at the level of protein abundance.
The purified mitochondria were also used to determine respiratory activities (Table 2). In the absence of calcium, external NADH oxidation was virtually identical in the two sets of mitochondria. However, calcium-dependent external NADH oxidation was almost twofold greater in mitochondria isolated from the ammonium treatment than the nitrate treatment. Similar results were observed using both O2 and the ubiquinone analogue decylubiquinone as the terminal electron acceptor (Table 2). The AOX capacity was almost threefold higher in mitochondria from the ammonium treatment as compared with the nitrate treatment (Table 2a), consistent with the whole seedling respiration measurements and the Western analysis (Figures 6 and 8). However, the AOX activity in the isolated mitochondria is generally low, indicating that the enzyme may have been partially inactivated during mitochondrial isolation (see Experimental procedures). Malate dehydrogenase activity was unaffected by the N source.
Table 2. NAD(P)H oxidation activities in intact mitochondria isolated from ammonium- or nitrate-supplied Arabidopsis seedlings
Specific activity [nmol NAD(P)H min−1 mg−1 protein]
All activities are given ± SD for two biological replicates, each consisting of 2–4 measurements. An asterisk denotes significant difference for P < 0.05.
30 ± 3
26 ± 4
70 ± 5
103 ± 8*
10 ± 3
24 ± 2*
+ N-propyl gallate
1.4 ± 0.3
0.5 ± 0.3
39 ± 1
77 ± 12*
8.2 ± 3.2
23 ± 1*
57 ± 8
46 ± 0.3
59 ± 3
100 ± 10*
13 ± 1
12 ± 2
55 ± 9
63 ± 6
(d) Malate dehydrogenase (NADH)
16 600 ± 1000
17 700 ± 4300
The specific increase in calcium-dependent external NADH oxidation by ammonium is fully consistent with the regulation of ndb4 (Figure 3), as NDB2–4 are predicted to be calcium-dependent external NADH dehydrogenases (Michalecka et al., 2003, 2004;Rasmusson et al., 2004). The nda2 gene, which responds to N (Figure 3), is believed to encode an internal NAD(P)H dehydrogenase (Moore et al., 2003; Rasmusson et al., 2004). However, we were unable to detect internal rotenone-insensitive NADH oxidation in the isolated mitochondria (results not shown). This may be due to the calcium-independent external NADH oxidation, which is high in these mitochondria (Table 2) and interferes with activity assays for the internal enzyme. External NADPH oxidation was not affected by the N source (Table 2c), which is consistent with the N-independent expression of the ndb1 gene, the Arabidopsis orthologue of the potato ndb1 gene that encodes an external NADPH dehydrogenase (Michalecka et al., 2004; Rasmusson et al., 2004).
Nitrogen fundamentally redirects plant metabolism, increasing carbon flow into organic acid synthesis to create 2-OG for ammonium assimilation, while simultaneously slowing the production of carbohydrates (Larsen et al., 1981; Paul et al., 1978; Scheible et al., 1997). The 2-OG utilized for N assimilation is synthesized via either two or three NAD+-reducing enzymes of the Krebs cycle (Hodges, 2002). In either case, NADH production in the mitochondrial matrix will increase substantially. We initiated the studies described above to determine whether the alternative pathways of the mitochondrial respiratory chain are upregulated by N to accommodate the increase in redox load accompanying 2-OG synthesis. Nitrate and ammonium have similar effects on organic acid synthesis and both require 2-OG for assimilation, so similar effects of the two N forms on the respiratory bypasses would imply such a function of the alternative pathways. However, nitrate and ammonium caused opposing changes in the expression of type II NAD(P)H dehydrogenases and AOXs, indicating that N regulation of the alternative pathways of the respiratory chain involves more factors than matrix NADH reduction level.
Our results demonstrate that three members of the AOX gene family (aox1a, aox1d, aox2) and two members of the type II NAD(P)H dehydrogenase gene family (ndb2, ndb4) are induced by ammonium, while genes encoding two AOXs (aox1a, aox1d) and one type II NAD(P)H dehydrogenase (nda2) are downregulated by nitrate. Ammonium- and nitrate-induced transcriptional changes are rapid and sustained, occur in both root and shoot tissues, and are accompanied by corresponding alterations in the capacity of AOXs and external type II NAD(P)H dehydrogenases. Thus, dehydrogenase and oxidase respiratory bypasses can be regulated in a coordinated manner, theoretically engaging/disengaging a complete non-proton pumping respiratory chain (see also Svensson and Rasmusson, 2001). Further, we have identified physiological factors controlling expression of the ndb genes, potentially providing insight into the so far mysterious physiological role(s) of external NAD(P)H dehydrogenases.
Recent work with a nitrate reductase double mutant of Arabidopsis has shown that nitrate per se is a potent signalling molecule, altering transcript levels of 595 genes (Wang et al., 2004). For the genes investigated, nitrate signalling appears to be wholly (aox1d, nda2) or partially (aox1a) mediated by the nitrate ion itself. Additionally, the induction of AOX capacity in seedlings transferred from nitrate to ammonium is relatively slow (Figure 6). However, this is consistent with the relatively slow transcript-level induction of aox1d and aox2 after a similar switch, with 8 h on ammonium having no effect or a much smaller inductive effect than a 56-h ammonium treatment (Figure 3). The slow disappearance of nitrate in the tissue (Figure 6) indicates that, during the initial phase after the medium switch, nitrate suppresses the expression of aox1d and potentially aox2 (Figure 3), though the latter was not nitrate suppressed in the N-stressed seedlings (Table 1). The studied ammonium-specific signalling pathways appear to be quite complex. As discussed previously, ammonium and nitrate differ in their effect on extracellular pH, with nitrate uptake causing soil alkalinization and ammonium uptake causing soil acidification (Crawford and Forde, 2002). Extracellular pH appears to have a large effect only on the expression of the aox2 and the ndb4 genes, both of which are upregulated under acidic conditions (and by ammonium) and preferentially expressed in root tissue. The provision of ammonium as an N source has been shown to cause a large, rapid decrease in rhizosphere pH [e.g. −1.7 units in oilseed rape (Muranyi et al., 1994) and −1.3 units in tea (Ruan et al., 2000)]. These changes are within the range of the pH shifts modulating expression of aox2 and ndb4. Because pH buffering of the seedling growth medium does not abolish (or in some cases even reduce) the inductive effects of ammonium, there is clearly also a pH-independent ammonium signalling pathway(s) controlling respiratory bypass gene expression.
Apoplastic pH in plants varies in response to several biotic and abiotic cues, and cytosolic pH changes have been investigated as a signalling factor (Felle, 2001), but regulation of gene expression by extracellular pH has not been analysed in plants. It has been reported that extracellular pH shifts do not affect the fluorescence signal of an intracellular pH probe, indicating that pH changes are not directly translocated over the plasma membrane (Gao et al., 2004). In fungi, several genes have been shown to be directly controlled by extracellular pH, and a signalling pathway involving a transmembrane receptor that may be responsive to changes in extracellular pH has been postulated (Penalva and Arst, 2004).
Nitrate assimilation has a much higher energetic cost than ammonium assimilation, requiring the additional oxidation of one cytosolic NADH and six reduced ferredoxins in the plastid. Considering that, after carbon fixation, nitrate assimilation can be the second largest sink for photosynthetic reductant (Noctor and Foyer, 1998), cellular redox status would be expected to be substantially different in nitrate-fed plants than in ammonium-fed plants. Changes in cell redox status, sensed by reactive oxygen species generation or alterations in the reduction state of ubiquinone, ascorbate, or pyridine nucleotide pools, can be important regulators of gene expression and may exert substantial control over the alternative respiratory pathways (Maxwell et al., 2002; Møller and Rasmusson, 1998; Noctor et al., 2004; Raghavendra and Padmasree, 2003). Thus, it is possible that redox inputs play a role in pH-independent ammonium signalling.
When seedlings grow on nitrate, the downregulation of a matrix-facing type II NAD(P)H dehydrogenase and several AOXs would effectively lower respiratory reoxidation of matrix NADH produced during 2-OG synthesis (Figure 9). With lower respiratory chain activity, more reductant (NADH) is available for export to the cytosol via, for example, the malate/oxaloacetate shuttle (Krömer, 1995). Because external (intermembrane space-facing) type II NAD(P)H dehydrogenases have a high Km compared with nitrate reductase (approximately 30 μm versus approximately 1.4 μm), they would not effectively compete for cytosolic NADH, allowing reductant exported from the mitochondria and plastids to be utilized for nitrate reduction (Krömer, 1995). In addition, all three of the respiratory bypass genes downregulated by nitrate (nda2, aox1a and aox1d) are relatively abundant in leaves (Figure 2), which correlates with the leaf being the primary site of nitrate assimilation (Foyer and Noctor, 2002).
In contrast, when ammonium is the N source, the alternative pathways of the respiratory chain are upregulated. In the absence of nitrate reduction, cytosolic NADH (arising from glycolysis and/or plastid redox export) may accumulate, allowing activation of the ammonium-induced external type II NAD(P)H dehydrogenases and AOX. Cytosolic redox equivalents can thus be shunted into the respiratory chain, stabilizing the cytoplasmic redox level (Gardeström et al., 2002; Krömer, 1995; Figure 9). The increased capacities of alternative respiratory pathways in ammonium-grown seedlings may also result in more respiratory oxidation of matrix NADH and less export of redox equivalents to the cytosol. Because ammonium assimilation occurs primarily in root tissues, the type II NAD(P)H dehydrogenases NDB2 and NDB4, which are somewhat more expressed in roots, and the root-specific AOX2 (all of which are ammonium-induced at the transcript level) are probably most physiologically relevant in ammonium metabolism under natural conditions.
Overall, the patterns of ammonium- and nitrate-mediated changes in the alternative respiratory pathways are entirely consistent with a role in the maintenance of whole cell redox homeostasis during N assimilation (Figure 9; see also Barneix et al., 1984; Lambers, 1980). Several studies over the past 20 years provide indirect or direct support for this hypothesis. At the genetic level, a recent report on the global effects of nitrate resupply to N-starved Arabidopsis seedlings has provided several interesting insights. Utilizing an exceptionally sensitive microarray set-up that allowed comparison of transcript abundance for five of the type II NAD(P)H dehydrogenase genes and two of the AOX genes, Scheible et al. (2004) found that expression of nda2, ndb2 and aox1a was suppressed in seedlings 3 h after nitrate resupply. These results are generally consistent with our data and the model described above (Figure 9). The effect of ammonium on the respiratory bypass genes has not previously been reported.
The switch of N source from nitrate to ammonium caused an increase in both AOX capacity and respiratory O2 consumption, which correlated with the disappearance of nitrate in the tissue (Figure 6). It has previously been demonstrated that the respiratory quotient (ratio of CO2 evolved to O2 consumed) is substantially lower with ammonium nutrition than nitrate nutrition in barley, wheat, maize and pea roots (Bloom et al., 1992; Cramer and Lewis, 1993; de Visser, 1985). This implies that more reductant is oxidized by the respiratory chain, as compared with Krebs cycle activity, under ammonium nutrition. These results support the hypothesis that the activity of the respiratory chain is altered by N supply, presumably to allow increased export of mitochondrial reductant to support cytosolic nitrate reduction (Bloom et al., 1992; Scheible et al., 2004). The central position of the alternative respiratory pathways in N-induced modulations of respiratory chain activity is suggested by our own work, but is also supported by the previous demonstration that root AOX capacity is substantially higher during growth with ammonium, rather than nitrate, as an N source (Blacquière and de Visser, 1984) as also suggested by Lambers (1980).
Finally, it is worthy of a note that N regulation of respiration appears to differ substantially between higher plants and algae. For example, expression of the Aox1 gene of Chlamydomonas reinhardtii is upregulated by nitrate and downregulated by ammonium (Baurain et al., 2003): the opposite of the trend we observed for AOXs in Arabidopsis. In addition, the effects of nitrate on CO2 release and O2 consumption differ substantially between algae and plants (Bloom et al., 1992; Weger and Turpin, 1989). Thus, because plants and algae appear to utilize quite divergent strategies for control of cellular energy/redox status (Bloom et al., 1992), it is unclear to what extent studies of N effects on algal respiration (e.g. Huppe and Turpin, 1994) can be interpreted in the context of higher plant respiratory metabolism.
Plant material and culture conditions
Arabidopsis (ecotype Columbia) seedlings were grown in shaking liquid cultures (125 rpm) at 22°C with a 16-h photoperiod (light intensity 40 μmol m−2 sec−1). The basic nutrient solution (termed ‘complete medium’ in the text) was as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mm KNO3, 1.25 mm KH2PO4 (pH5.6), 1 mm MgSO4, 1 mm Ca(NO3)2, 25 μm Fe-EDTA and the reported micronutrient mix at 1× concentration], supplemented with 2% (w/v) sucrose. Approximately 100 surface-sterilized seeds were sown in 100 ml of sterile complete medium in a 250-ml flask and grown for 8 or 9 days.
For N starvation and resupply experiments, 9-day-old seedlings were rinsed in sterile water and transferred to a modified nutrient medium lacking N [KNO3 replaced with KCl and Ca(NO3)2 replaced with CaCl2] for 3 days. The seedlings were then resupplied with 9 mm KNO3, 4.5 mm (NH4)2SO4, or 4.5 mm K2SO4 and harvested after 2, 8, or 24 h. These concentrations of nitrate and ammonium are low compared with N levels in most plant tissue culture media (e.g. Murashige and Skoog, Gamborg B5) but on the high end of levels in agricultural soils (Britto et al., 2001; Crawford and Forde, 2002). For experiments in which the N source was alternated between nitrate and ammonium, 8-day-old seedlings were rinsed in sterile water and transferred to the modified nutrient medium (as above) supplemented with either 4.5 mm KNO3 or 2.25 mm (NH4)2SO4 for 2 days. The seedlings were then rinsed again and transferred to the same medium (nitrate–nitrate and ammonium–ammonium controls) or the medium with the alternative N source (nitrate–ammonium and ammonium–nitrate switch). Seedlings were harvested 8 h after medium change. No symptoms of ammonium toxicity [e.g. interveinal chlorosis and marginal necrosis of leaves, or browning of roots (Walch-Liu et al., 2000)] were seen upon 2–48 h exposure to ammonium nutrition.
RNA isolation, cDNA synthesis and real-time PCR analysis
RNA isolation, analysis and spectrophotometric quantitation were performed as in Escobar et al. (2004). cDNA was synthesized from 1 μg of total RNA using the RevertAid H Minus First Strand cDNA synthesis Kit (Fermentas, St Leon-Rot, Germany) according to the manufacturers specifications, except for the addition of a final RNAse H digestion step (2U RNAseH/reaction with incubation at 37°C for 20 min). First strand cDNA was diluted fivefold in 10 mm Tris-HCl (pH 8.0) for use in real-time PCR reactions.
Real-time PCR was performed on a Rotor Gene 2072 Real-Time Cycler (Corbett Research, Sydney, Australia) as described previously (Escobar et al., 2004), except for the use of a hot start DNA polymerase (Platinum Taq; Invitrogen, Carlsbad, CA, USA) for the amplification of transcripts that in some treatments were difficult to detect (ndb4 and aox2). Relative transcript abundance was quantified using comparative quantitation analysis settings (Rotor Gene software version 4.6). Note that the values presented from real-time analysis are relative and transcript-specific and thus not comparable between the different genes. All utilized primer pairs have been described previously (Escobar et al., 2004; Michalecka et al., 2003) except ucp4 (At1g14140; 5′-CTC CGG CGA TTA TCA GAC AC-3′, 5′-TGG ACT AGC CAC TAC CTG AGC-3′) and nia2 (At1g37130; 5′-GAT CAT CAT CCC CGG TTT C-3′, 5′-CTG GCT TAT ACC ACC AAC CTT C-3′). At least one primer in each pair spans an exon–exon border, precluding amplification of genomic DNA. All real-time PCR data are presented ±SD for at least two biological replicates, with at least two independent PCR reactions run for each biological replicate. Statistical tests were done by one-way analysis of variance using spss 11.0.2 software (SPSS Inc., Chicago., IL, USA).
For mitochondrial isolation, 8-day-old seedlings were rinsed in sterile water and transferred to modified nutrient medium supplemented with either 4.5 mm KNO3 or 2.25 mm (NH4)2SO4 for a total of 2 days, with daily transfer to fresh medium. In initial experiments, mitochondria were isolated using the method of Kruft et al. (2001). However, no AOX activity was detected in these mitochondria, though they were intact as seen from malate dehydrogenase latency (data not shown). This is consistent with previously reported difficulties in purifying functional mitochondria from Arabidopsis tissues (except cell suspension cultures; Hausmann et al., 2003). Therefore, we modified the homogenization buffer to include 100 mm ascorbate, which inhibits production of reactive compounds by myrosinase and has been used for isolating import-competent Arabidopsis chloroplasts (Schulz et al., 2004).
Whole seedlings (10 g) were homogenized in 240 ml of modified extraction buffer [0.25 m sucrose, 1.5 mm EDTA, 100 mm ascorbic acid, 15 mm 4-morpholine propanesulfonic acid (MOPs), pH 7.4 (KOH), 0.4% (w/v) delipidated bovine serum albumin, 0.6% (w/v) polyvinyl pyrrolidone-40 and 10 mm dithiothreitol] using a mortar and pestle. The homogenate was filtered through a 50-μm nylon mesh and cellular debris was pelleted by one centrifugation at 2200 g for 5 min. The supernatant was centrifuged at 17 000 g for 10 min, and the pellet of crude mitochondria was resuspended in gradient buffer, homogenized, transferred to a microtube and centrifuged at 2200 g for 2 min. The supernatant was then loaded directly on a step gradient of 2.5 ml 45% and 6.5 ml 27% Percoll and centrifuged at 20 000 g for 20 min. Gradient buffer, wash buffer and all additional steps were as previously described (Kruft et al., 2001). Potato leaf mitochondria were isolated as previously described (Svensson and Rasmusson, 2001). Total protein concentration in mitochondrial preparations was determined by the bicinchoninic acid protein assay (Sigma-Aldrich, St. Louis, MO, USA). Chlorophyll concentration was determined by acetone extraction (Arnon, 1949).
Mitochondrial protein (20 μg/lane) was resolved in 10% SDS-PAGE gels, and wet-transferred to nitrocellulose membranes in 10 mm 3-(cyclohexylamino)-1-propanesulfonic acid, pH 11 (NaOH), 20% (v/v) methanol (Moos et al., 1988). Molecular masses were determined with pre-stained markers (Bio-Rad, Hercules, CA, USA) and immunodecoration was made as in Michalecka et al (2004).
Malate dehydrogenase activity and latency was determined spectrophotometrically (Møller et al., 1987) on a Shimadzu UV-160A spectrophotometer (Kyoto, Japan).
Oxidation of NAD(P)H in intact mitochondria was measured with an Aminco DW2a dual wavelength spectrophotometer at 340–400 nm. For quantifying NADH oxidation to O2, the reaction medium (Medium A) contained 20 mm HEPES, 100 mm KCl, 100 mm sucrose, 2.5 mm MgCl2 and 0.5 mm EGTA at pH 7.8 (KOH). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.4 μm) and pyruvate (5 mm) were added to the assay mixture. To certify maximum AOX activity, mitochondria were pre-incubated for 10 min in 5 mm dithiothreitol in a small volume, leading to a concentration of 20 μm being present in the assay. The reaction was started by addition of 100 μm NADH, followed by 1 mm CaCl2. To inhibit bc1 complex activity for determination of AOX capacity, 0.375 μm antimycin A was added. Alternative oxidase was inhibited by addition of 50 μmn-propyl gallate.
External NADH oxidation to decylubiquinone was measured in Medium A, supplemented with 0.375 μm antimycin A, 40 μm decylubiquinone and 0.1 mm NADH and in the presence or absence of 1 mm CaCl2. Internal type II NADH dehydrogenase activity was measured in the presence of 15 μg ml−1 alamethicin and 15 μm rotenone, as described by Johansson et al. (2004), with pyruvate and dithiothreitol added as for external NADH to O2 activity. External NADPH to decylubiquinone activity was assayed as previously described (Johansson et al., 2004).
Coupling of respiration to oxidative phosphorylation was measured in a medium containing 0.3 m sucrose, 10 mm MOPS, 1 mm KH2PO4, pH 7.2 (KOH), 0.1% defatted bovine serum albumine, 2.5 mm MgCl2, 0.2 mm CaCl2 and 0.1 mm ATP. To minimize AOX activity, pyruvate and dithiothreitol was omitted.
For determinations of O2 consumption activities of intact Arabidopsis seedlings, approximately 15 seedlings were grown and treated in 50-ml cultures. Media were not buffered, but were changed daily during the treatments, thereby allowing for the fluctuations in pH associated with each form of N nutrition (Hinsinger et al., 2003; Ruan et al., 2000), while avoiding extended incubation below pH 4.5 or above pH 6.5. Oxygen consumption in seedlings (125–325 mg fresh weight) was measured in respective growth media at 25°C in the dark, using a 5 ml O2 electrode (Rank Brothers, Cambridge, UK). Oxygen uptake was measured immediately after transferring seedlings to the reaction vessel, 5 min after the addition of KCN (1 mm) and again 5 min after the addition of n-propyl gallate (0.1 mm), with re-oxygenation of the medium during inhibitor incubations. The AOX capacity was calculated as the rate with KCN minus the rate with KCN and n-propyl gallate. The tissue total respiration rate was calculated as the total O2 uptake minus the rate in the presence of both inhibitors (10–15% of the total). Addition of n-propyl gallate alone slightly increased respiration in the seedlings, presumably due to the activation of KCN-sensitive peroxidases (see Møller et al., 1988), precluding the determination of cytochrome pathway capacity. For nitrate determination, the seedlings were rinsed in distilled water and analysed according to Cataldo et al. (1975).
This work was supported by the Swedish Research Council and Carl Tesdorpfs Stiftelse. We are grateful to Dr Alexandra Mant, The Royal Veterinary and Agricultural University, Denmark for valuable suggestions on organellar isolation and to Drs Jean-Michel Grienenberger (Institute de Biologie Moléculaire des Plantes-Centré National de la Recherche scientifique, Strasbourg, France), Tadashi Asahi (Nagoya University, Japan) and Thomas E. Elthon (University of Nebraska-Lincoln, Lincoln, NE, USA) for the generous donation of antibodies. MAE acknowledges a fellowship from the Wenner-Gren Foundations.