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Plant, Cell and Environment (2012)
doi: 10.1111/j.1365-3040.2012.02559.x
The role of mitochondria in leaf nitrogen metabolism
pce_2559
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BOZ˙ENA SZAL & ANNA PODGÓRSKA
Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland
ABSTRACT For optimal plant growth and development, cellular nitrogen (N) metabolism must be closely coordinated with other metabolic pathways, and mitochondria are thought to play a central role in this process. Recent studies using genetically modified plants have provided insight into the role of mitochondria in N metabolism. Mitochondrial metabolism is linked with N assimilation by amino acid, carbon (C) and redox metabolism. Mitochondria are not only an important source of C skeletons for N incorporation, they also produce other necessary metabolites and energy used in N remobilization processes. Nitric oxide of mitochondrial origin regulates respiration and influences primary N metabolism. Here, we discuss the changes in mitochondrial metabolism during ammonium or nitrate nutrition and under low N conditions. We also describe the involvement of mitochondria in the redistribution of N during senescence. The aim of this review was to demonstrate the role of mitochondria as an integration point of N cellular metabolism. Key-words: alternative oxidase; amino acid metabolism in mitochondria; nitrate and ammonium nutrition; reassimilation of ammonia.
INTRODUCTION In plants, similarly to other living organisms, most primary and many secondary metabolites contain nitrogen (N) in their structures. Low N availability or improper N use efficiency may limit plant biomass and subsequent food production. Therefore, the understanding of details of N cellular metabolism is of great importance, both in plant physiology and in biotechnology and agriculture. Plant mitochondria may be recognized as an integration point of cellular metabolism and signalling (Sweetlove et al. 2007). The unique functions of plant mitochondria are due to the presence of alternative respiratory chain components that bypass the complex I or cytochrome pathway, different bypasses of the tricarboxylic acid (TCA) cycle and plantspecific metabolite exchangers between mitochondria and the cytosol (Møller 2001; Plaxton & Podestá 2006; Sweetlove et al. 2007; Rasmusson, Geisler & Møller 2008; Linka & Weber 2010). Mitochondrial N metabolism is best understood in the context of supplying C skeletons for N incorporation; Correspondence: B. Szal. E-mail:
[email protected] © 2012 Blackwell Publishing Ltd
however, it is now apparent that mitochondria are engaged in many other processes and pathways. Reductants that originate from mitochondrial photorespiratory activity are crucial for the primary assimilation of N. Some recently published results indicate that reassimilation of photorespiratory NH3 indeed may occur within mitochondria. Due to their plasticity, plant mitochondria may also regulate the cellular redox state under ammonium nutrition or C/N ratio under a suboptimal N supply. In plant cells, specific pathways in the mitochondrial electron transport chain (mtETC) and the Krebs cycle enable the use of amino acids as an alternative source of energy, which is especially important under stressful conditions. Novel findings support the role of mitochondria in senescence-associated nutrient remobilization or nitric oxide (NO) production. The aim of our review was to emphasize the central role of mitochondria in the anabolic, catabolic and signalling pathways of N metabolism.
MITOCHONDRIA AND PRIMARY ASSIMILATION OF N NO3- is the most common N source for plants. Nitrate (NO3-) is transported across the plasma membrane concomitantly with a proton (H+), and the symport across membranes is driven by pH gradients. The depolarization is counteracted by the plasma membrane H+-ATPase and depends on ATP generated mainly in the mitochondria. The mechanisms of N transport across the membranes were reviewed recently by Dechorgnat et al. (2011) and Kraiser et al. (2011).When plants are growing with a supply of NO3-, the first step in the reduction process is localized in the cytosol and the conversion of NO3- to nitrite (catalysed by NO3- reductase, NR) requires reducing equivalents. NADH level may be a limiting factor for nitrate reduction since even under photorespiratory conditions the concentration of cytosolic NADH is about 0.3–0.7 mm (Heineke et al. 1991) what is below the Km of NR (7 mm) (Kaiser et al. 2000). In addition to cytosolic-localized reactions that reduce nicotinamide adenine dinucleotide (NAD+) to NADH (e.g. glycolysis), a major part of the reductants is of chloroplast or mitochondrial origin. Under steady-state photosynthesis, up to 50% of NADH produced in mitochondria [mainly from the glycine (Gly) decarboxylase complex activity] may be exported to the cytosol (Fig. 1; Krömer & Heldt 1991; Krömer 1995). The mitochondrial ‘malate shuttle’ driven by photorespiratory metabolism increases the NADH/NAD ratio in the cytosol and thereby 1
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PEROXISOME
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Figure 1. Simplified scheme showing involvement of leaf mitochondria in NO3- assimilation. Mitochondria provide 2-oxoglutarate (2-OG) for chloroplast-localized glutamine synthetase–glutamine:2-OG aminotransferase (GS-GOGAT) cycle and reductants used by cytosolic NO3- reductase (NR).
provides reductants for NR activity in C3 plants (Rachmilevitch, Cousins & Bloom 2004; Bloom et al. 2010). The experimental data concerning differences in cytosolic NADH concentration in photorespiratory versus nonphotorespiratory conditions are scarce (e.g. Igamberdiev et al. 2001). But, if primary NO3- assimilation in great part depends on photorespiration an attempt to eliminate the oxygenation reaction of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) may consequently lead to N deficiency in plants (Rachmilevitch et al. 2004; Bloom et al. 2010). In chloroplasts, for the proper action of the glutamine (Gln) synthetase–Gln:2-oxoglutarate (2-OG) aminotransferase (GS-GOGAT) cycle, there must be constant access to C skeletons (2-OG) and the mitochondria are one of the sources of those needed for this cycle (Tcherkez et al. 2009; Gauthier et al. 2010). It was proven that during light conditions, remobilization of night-stored organic acids plays a significant role in providing 2-OG for Glu synthesis (Gauthier et al. 2010). Under light conditions, partial operation of the Krebs cycle also takes place (Hurry et al. 2005; Sweetlove et al. 2010) despite light-dependent inhibition of the mitochondrial pyruvate (Pyr) dehydrogenase complex (PDC; Pärnik & Keerberg 1995; Tovar-Méndez, Miernyk & Randall 2003; Tcherkez et al. 2005) and redox-dependent inhibition of other dehydrogenases of the Krebs cycle (Igamberdiev & Gardeström 2003; Tovar-Méndez et al. 2003; Balmer et al. 2004; Bykova et al. 2005; Tcherkez et al. 2005; Araújo et al. 2008). It remains under debate which organic acid is exported from the mitochondria (Foyer, Noctor & Hodges 2011). 2-OG may be withdrawn from the
TCA cycle and exported to the cytosol through the di/tricarboxylate transporter (Fig. 1; Hodges 2002; Picault et al. 2002, 2004; Haferkamp 2007; Noguchi & Yoshida 2008, Linka & Weber 2010). Alternatively, citrate or isocitrate can be exported from mitochondria (by the same transporter) and converted to 2-OG in the cytosol (Lancien, Gadal & Hodges 2000; Hodges 2002; Igamberdiev & Gardeström 2003). Mitochondrial NAD- or NADP-isocitrate dehydrogenase (-IDH) would be involved in 2-OG production when 2-OG is withdrawn, whereas cytosolic NADP-IDH is involved when citrate or isocitrate is transported from the mitochondria. Nuclear magnetic resonance (NMR) studies have indicated that the main mitochondrial product in illuminated leaves is citrate (Gout et al. 1993); however, recent results obtained using transgenic plants suggest that no specific IDH isoform is involved in the production of 2-OG for N assimilation and both the above reactions might take place (Foyer et al. 2011 and references therein).
REASSIMILATION OF AMMONIA GENERATED IN THE PHOTORESPIRATORY PATHWAY The photorespiratory pathway involves several enzymatic reactions localized in the chloroplasts, mitochondria, peroxisomes and cytosol (Leegood et al. 1995; Gardeström, Igamberdiev & Raghavendra 2002; Timm et al. 2008, 2011; Bauwe, Hagemann & Fernie 2010; Peterhansel & Maurino 2011). In mitochondria, during reactions catalysed by Gly decarboxylase complex (GDC) and Ser hydroxymethyltransferase (SHMT) NADH and ammonia (NH3) are released. NH3 is a valuable form of reduced N and has to be © 2012 Blackwell Publishing Ltd, Plant, Cell and Environment
Mitochondria and N metabolism
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GS-GOGAT cycle (Fd-GOGAT) is also targeted to both the mitochondria and the chloroplasts (Jamai et al. 2009), indicates that NH3 reassimilation process might effectively work both in chloroplasts and in mitochondria. Until now, no source of electrons for mitochondrial Fd-GOGAT was found and, instead, a regulatory role of mtFd-GOGAT was proposed. It is most probable that mtFd-GOGAT is required for optimal SHMT operation (Jamai et al. 2009); however, Gln generated in the matrix because of mtGS2 activity might be exported from the mitochondria simultaneously with importation of Glu to maintain the stoichiometry of the reaction (Fig. 2b; Linka & Weber 2005). Alternatively, it was proposed that mitochondrial NH3 assimilation is coupled with the transfer of carbon dioxide in a citrulline–ornithine shuttle between chloroplasts and mitochondria (Fig. 2c; Taira et al. 2004). This pathway consumes large amounts of ATP because the mitochondrial carbamoyl phosphate synthetase (CPS) activity allows direct regeneration of the NH3 acceptor (Glu; Fig. 2c) within the mitochondrial matrix. The existence of this pathway was proved by Taira et al. (2004), who demonstrated that isolated mitochondria are able to catalyse Gly-dependent conversion of ornithine to citrulline. NH3 reassimilation in mitochondria requires further examination, and recent reports indicate that the photorespiratory pathway may be more complicated than previously anticipated.
Orn OCT
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Figure 2. The pathways of reassimilation of ammonia produced by Gly decarboxylase complex (GDC). (a) Simple diffusion of NH4+ from mitochondria to chloroplast and then ammonium assimilation in glutamine synthetase–glutamine:2-OG aminotransferase (GS-GOGAT) cycle. (b) Assimilation of ammonium in mitochondria by mtGS2 and transport of Gln to chloroplasts. (c) Citrulline-ornithine shuttle enabling parallel transport of ammonium and CO2 from mitochondria to chloroplasts. Dotted arrows represent possible processes.
reassimilated. The reassimilation process exceeds primary assimilation of N by one order of magnitude (Keys et al. 1978). For decades, it was believed that NH4+ simply diffuses into the chloroplast, where it is used in the GS-GOGAT cycle (Fig. 2a); however, some new experimental results indicate other possibilities. Taira et al. (2004) found that GS2 is a dual-targeted peptide. Targeting the GS2 isoform to plastids and mitochondria suggests that incorporation of NH4+ into Gln structure might take place in both organelles. Surprisingly, when Taira et al. (2004) compared GS activity in purified chloroplasts and mitochondria, the later was about 4- to 10-fold higher than former. Moreover, organellar GS activity in both chloroplasts and mitochondria was induced in photorespiratory conditions (Taira et al. 2004). Together with the finding that the second enzyme of the © 2012 Blackwell Publishing Ltd, Plant, Cell and Environment
MITOCHONDRIALLY LOCALIZED AMINO ACID METABOLISM The anabolic pathways of amino acids (AAs), including those mitochondrially localized such as cysteine, were described in detail by Morot-Gaudry, Job & Lea (2001) and Sweetlove et al. (2007 and references therein); therefore, this review will be focused on AA catabolism linked with the Krebs cycle or mtETC function. The most important mitochondrial AA catabolic pathway is Gly decarboxylation under photorespiratory conditions (Gardeström et al. 2002; Raghavendra & Padmasree 2003). The impact of NADH produced by GDC on the primary N assimilation process and the proposed pathways involved in reassimilation of NH4+ have already been discussed. Another important mitochondrial AA catabolic pathway is the reaction catalysed by Glu dehydrogenase (GDH) (Fig. 3). GDH may theoretically catalyse the amination reaction of 2-OG and deamination of Glu (Aubert et al. 2001; Dubois et al. 2003). The possible involvement of GDH in N assimilation under conditions of inorganic N excess was shown by MeloOliveira, Oliveira & Coruzzi (1996). However, it was experimentally documented that the main function of GDH in planta is deamination of Glu into 2-OG, which is then incorporated into the Krebs cycle (Miflin & Habash 2002; Dubois et al. 2003; Masclaux-Daubresse et al. 2006). The 2-OG flow from Glu oxidation is especially important under limited C conditions (Robinson, Stewart & Phillips 1992; Masclaux-Daubresse et al. 2006 and references therein).
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Figure 3. Mitochondrial pathways of amino acid (AA) catabolism and its relation with the tricarboxylic acid (TCA) cycle or mitochondrial electron transport chain (mtETC).
Mitochondria are also involved in the catabolism of Glu through the g-aminobutyrate (GABA) shunt (Shelp, Bown & McLean 1999; Bouché & Fromm 2004; Fait et al. 2008). GABA metabolism is associated with regulation of cytosolic pH, protection against oxidative stress or insects and osmoregulation (Bouché & Fromm 2004). Besides, GABA is proposed to be a signalling molecule involved in the maintenance of C/N homeostasis and is produced under both non-stress and stress conditions (Tcherkez et al. 2009). Within the GABA shunt, Glu in the cytosol is decarboxylated to GABA. It is then transported into the mitochondria and the matrix through the reactions catalysed by GABA transaminase (GABA-T), and succinic semialdehyde dehydrogenase (SSADH) is further metabolized to succinate, which enters the Krebs cycle (Fig. 3). Mitochondrial GABA-T can use either Pyr or 2-OG as an amino acceptor and to catalyse the conversion of GABA to SSA leading to the formation of alanine (Ala) or Glu, respectively (Van Cauwenberghe & Shelp 1999; Bouché & Fromm 2004; Fait et al. 2008). In addition to SSADH activity, SSA may be
converted to 4-hydroxybutyrate (GHB) by GHB dehydrogenase and further metabolized to acetyl-coenzyme A (CoA; Fait et al. 2008). Fait et al. (2008) have suggested that a GABA shunt may even be approved as an integral part of the TCA cycle because of the many routes of intermediate incorporation into the TCA cycle and positive correlation of the expression of the GABA shunt regulatory enzymes (e.g. SSADH) with genes associated with the TCA cycle. Proline (Pro) accumulation is a well-known response of plants to stress conditions resulting from its increased synthesis concomitantly with its inhibited degradation. During recovery from stress, accumulated Pro is rapidly oxidized and serves as source of N, reducing equivalents and energy. Pro is oxidized in the mitochondria to Glu by sequential action of Pro dehydrogenase (ProDH) and D1-pyrroline-5carboxylate dehydrogenase (P5CDH; Fig. 3; Mani et al. 2002; Deuschle et al. 2004). ProDH is bound to the inner mitochondrial membrane with its active site facing the matrix, whereas P5CDH is located inside the mitochondrial matrix (Elthon & Stewart 1981). NADH produced by © 2012 Blackwell Publishing Ltd, Plant, Cell and Environment
Mitochondria and N metabolism P5CDH is used in mtETC and Glu after deamination to 2-OG may be incorporated into the Krebs cycle (Plaxton & Podestá 2006). In barley leaves recovering from stress, the oxidation of Pro accounted for up 20% of respiration (Steward & Voetberg 1985). Plant mitochondria also contain different specific dehydrogenase complexes that are capable of oxidizing branched chain AAs [e.g. valine (Val), leucine (Leu) and isoleucine (Ile)] (Binder, Knill & Schuster 2007). In the first step of the mitochondrial-localized branched-chain AA catabolic pathway, Val, Leu and Ile are transaminated to their respective a-keto acid by the branched-chain AA transaminase (BCAT; Fig. 3; Diebold et al. 2002; Maloney et al. 2010).The a-keto acids are further decarboxylated and esterified to CoA by the branched-chain keto acid dehydrogenase complex (BCKDC) (Taylor et al. 2004). Mitochondrial localization of BCKDC was confirmed by Fujiki et al. (2000). The CoA esters generated by BCKDC are then oxidized by acyl-CoA dehydrogenases, and electrons are delivered through electron transfer flavoprotein/electron transfer flavoprotein:ubiquinone oxidoreductase (ETF/ ETFQO) system directly to mtETC (Taylor et al. 2004; Ishizaki et al. 2005, 2006). Arabidopsis mitochondria contain acyl-CoA dehydrogenase (isovaleryl-CoA dedydrogenase, IVD) showing activity towards both isovaleryl-CoA (from Leu) and isobutyryl-CoA (from Val) linked with the ETF/ ETFQO system (Däschner et al. 1999; Däschner, Couée & Binder 2001; Araújo et al. 2010). Subsequent steps along the catabolic pathways lead to the formation of propionyl-CoA (Val catabolism), propionyl-CoA and acetyl-CoA (Ile), and acetyl-CoA and acetoacetate in Leu metabolism (Taylor et al. 2004; Kochevenko & Fernie 2011); however, only in Leu are all reactions involved in the catabolic pathway localized in the mitochondria (Anderson et al. 1998; Taylor et al. 2004). The ETF/ETFQO system is also involved in the transport of electrons from 2-hydroxyglutarate (through 2-hydroxyglutarate dehydrogenase), which is an intermediate in lysine (Lys) metabolism (Araújo et al. 2010). The branched-chain AA catabolism is induced during C starvation when AAs may be used as alternative substrates for respiration (Fujiki et al. 2001, 2002). On the other hand, the regulation of branched-chain AA oxidation may also be a detoxification mechanism allowing a pool of branchedchain AAs to be maintained for protein synthesis while preventing their build-up to toxic levels (Taylor et al. 2004).
MITOCHONDRIAL ACTIVITY UNDER LOW N SUPPLY
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Scheible, Krapp & Stitt 2000; Noguchi & Terashima 2006). Low N availability also affects mtETC function. A decrease in the cytochrome pathway and an increase in alternative pathway activities were found simultaneously with a decrease of type II NAD(P)H dehydrogenases (NDin/ex; Sieger et al. 2005; Noguchi & Terashima 2006). Under low N, when demands for ATP is low because of slow protein turnover and lower phloem loading, the activity of the cytochrome pathway is restricted and up-regulation of alternative oxidase (AOX) allows efficient consumption of excess carbohydrates (Noguchi & Terashima 2006 and references therein). On the other hand, the increase of the level of cytosolic GS1, which was documented under low N conditions (Lothier et al. 2011), may create a high demand for mitochondrial ATP; therefore, the role of the cytochrome pathway could also be important.
CHANGES IN MITOCHONDRIAL METABOLISM INDUCED BY NO3- OR NH4+ SUPPLY The form in which N is supplied exerts specific influence on whole plant metabolism. When plants grow on NO3-, the two-step reduction reaction from NO3- to NH4+ requires eight electrons. The NO3- assimilation process occurs mainly in the leaves. Because part of NH4+ is directly incorporated into the AAs in root plastids, N is translocated to the leaves with the xylem sap in the form of AAs or NH4+. The uptake of different forms of N alters reductants and the energy status in leaf cells (Fig. 4). It was suggested that under NH4+ nutrition, plants must export excess NAD(P)H from the chloroplasts to prevent photoinhibition (Guo et al. 2005). Different electron demand for NO3- reduction and NH4+ assimilation may affect not only cytosolic and plastidic metabolism but also pathways located in the mitochondria. It was proposed that during NH4+ assimilation, both the
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Restricted N availability results in elevated carbohydrate levels in plant tissue (Paul & Driscoll 1997; Sieger et al. 2005). Under N-limited conditions, mitochondrial respiration allows consumption of excess C so that the appropriate C/N ratio may be maintained. An increase in the activity of Krebs cycle enzymes [NAD- and NADP-IDH, fumarase (Fum), citrate synthase (CS)] and NAD-malic enzyme (NAD-ME) was observed in different plant species in response to low N conditions (Makino & Osmond 1991; © 2012 Blackwell Publishing Ltd, Plant, Cell and Environment
REDUCTANTS MITOCHONDRION
REDUCTANTS MITOCHONDRION
Figure 4. Scheme illustrating the differences in redox status of the leaf cells under nitrate (a) and ammonium nutrition (b). In response to ammonium nutrition, cytosolic and mitochondrial redox state greatly increases. High influx of electrons to mitochondrial electron transport chain (mtETC) during ammonium supply may lead to increase in mitochondrial reactive oxygen species (mtROS) production.
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TCA cycle and mtETC activities increase (Weger & Turpin 1989). This increase results from greater demand for intermediates of the TCA cycle (2-OG or citrate) as C skeletons and the necessity for simultaneous oxidation of the excess reductants not used in the reactions catalysed by NR and nitrite reductase (NiR). During NO3- nutrition, when reductant export from the mitochondria is needed to support reduction reactions, the TCA cycle activity is strongly increased, also providing C skeletons for the GS-GOGAT cycle, and mtETC activity is unaltered. The effect of different levels of N nutrition on respiratory activity has been measured in various plant species; stimulation of respiratory activity by NH4+ was found (e.g. in barley, Arabidopsis thaliana, spinach, pea and French bean plants; Rigano et al. 1996; Lasa et al. 2002; Brück & Guo 2006; Escobar, Geisler & Rasmusson 2006). Furthermore, it was suggested that involvement of the cytochrome and an alternative pathway in total respiration depend on the available (assimilated) form of N. It is known that alternative pathway lowers the efficiency of mitochondrial ATP production but allows it to oxidize the excess cellular reductants, enabling the growth of plants in stress conditions (Purvis & Shewfeldt 1993; Møller 2001; Rasmusson et al. 2008); therefore, AOX might play an important role in dissipating excess redox equivalents during NH4+ supply. Indeed, Barneix, Breteler & van de Geijn (1984) and Blacquière & de Visser (1984) found an increase of AOX capacity during NH4+ nutrition. In fact, an increased AOX transcript level (mainly aox1a, aox1d and aox2), together with higher AOX capacity, was documented in NH4+-supplied Arabidopsis plants (Escobar et al. 2006). Contrary to those observations, the increase of cytochrome c oxidase (COX) activity under NH4+ supply was recently found by Hachiya et al. (2010), who suggested that this might be a result of the energy demand in NH4+-grown plants caused by the higher H+ATPase activity preventing cytosolic acidification. However, as acidification of cytosol during NH4+ supply was previously ruled out (Britto & Kronzucker 2005), the potential role of COX needs further examination. When comparing reductants and energy demand under NO3- with NH4+ nutrition, one may expect that the favourable N form for plants is NH4+. Surprisingly, when NH4+ is supplied as an exclusive N source, it causes symptoms of toxicity such as growth inhibition or chlorosis, called ‘NH4+ syndrome’ (Hoffmann et al. 2007). It was proposed that NH4+ syndrome was caused by oxidative stress (Zhu et al. 2000). According to Guo et al. (2005), under NH4+ nutrition, increased respiratory activity in NH4+-supplied plants may cause higher mitochondrial reactive oxygen species (mtROS) formation leading to oxidative stress.We observed ROS accumulation in mitochondrial membranes during NH4+ nutrition detected cytochemically as cerium perhydroxide precipitates (A. Podgórska, B. Szal, unpublished results), but this hypothesis of Guo et al. (2005) requires verification. We suggest that under NH4+ supply, mtROS are mainly involved in retrograde signal transduction, which is similar to that proposed under other stress conditions (Rhoads et al. 2006; Noctor, De Paepe & Foyer 2007).
THE INVOLVEMENT OF MITOCHONDRIAL METABOLISM IN N REALLOCATION DURING SENESCENCE Senescence-associated nutrient remobilization is common in all plant species (Himelblau & Amasino 2001 and references therein). In this regulated process, N and other nutrients are reallocated from old senescing leaves towards growing organs (young leaves, seeds, etc.). It was documented that in mesophyll cells, chloroplasts are dismantled in the early phase of senescence, while mitochondria remain functional until the late phase (Hörtensteiner & Feller 2002 and references therein; Keech et al. 2007). In fact, the up-regulation of genes associated with mitochondrial metabolism was found in senescing poplar leaves (Bhalerao et al. 2003). Recently, it was documented that the respiratory activity of mitochondria isolated from senescing Arabidopsis leaves was high with all tested substrates (malate, NADH, succinate and Gly; Keech et al. 2007). The high respiratory rate with Gly is surprising because the photorespiratory pathway in senescing leaves is rather low as a result of Rubisco degradation; however, Gly and other AAs may be derived from proteolysis.The importance of branched-chain AAs as respiratory substrates during senescence was demonstrated using ETFQO mutants (Ishizaki et al. 2005, 2006). Keech et al. (2007) suggested that the main role of mitochondria up to the late phase of senescence is to supply ATP and C skeletons needed for efficient redistribution of N.
THE EFFECT OF IMPAIRMENT OF MITOCHONDRIAL MATRIX-LOCALIZED ENZYMES ON CELLULAR N METABOLISM N is an essential component of most of the primary and many secondary metabolites; therefore, the term ‘changes in N metabolism’ may include a wide spectrum of processes. In this review, we focus on the role of mitochondria in NO3assimilation and the free AA cellular pool. In general, the impairment of the TCA cycle activity leads to an increase in NO3- concentration (Fig. 5), which
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Figure 5. The changes in nitrate, total amino acid (AA) levels, and individual AA concentration resulting from dysfunction of Krebs cycle enzymes. Data from (1) Sienkiewicz-Porzucek et al. (2008); (2) Carrari et al. (2003); (3) Lemaitre et al. (2007); (4) Sienkiewicz-Porzucek et al. (2010); (5) Araújo et al. (2008); (6) Studart-Guimarães et al. (2007); (7) Araújo et al. (2011); (8) Nunes-Nesi et al. (2007); (9) Nunes-Nesi et al. (2005); (10) Tomaz et al. (2010); (*) both mt and cytosolic isoform Aco were impaired (**) data for tuber discs. © 2012 Blackwell Publishing Ltd, Plant, Cell and Environment
Mitochondria and N metabolism may be a result of a restriction in the mitochondrial origin reductants used by NR; however, it is hard to draw a conclusion on the effect of the impaired activity of the TCA cycle on the AA content in leaves (Fig. 5). The spectrum of changes, which was observed to be very wide, and sometimes the dysfunction of the specific enzyme, may cause a different effect. For example, compared to wild-type plants, the increased Glu level was in SDH14 and SDH43 lines but in SDH52 transgenic line, the Glu level was unchanged (Araújo et al. 2011). To some extent, such diversity may be a result of the fact that in most cases, the inhibition of specific mitochondrial enzyme activity is not complete (papers by A.R. Fernie’s group, reviewed by Nunes-Nesi, Araújo & Fernie 2011). Moreover, nearly all of the studied Krebs’s cycle mutants or transgenes were derived from a wild-type tomato and some of the changes may not be universal within the entire plant kingdom (e.g. an opposite effect concerning the influence of mtMDH dysfunction on the photorespiratory pathway was observed in the tomato; increased photorespiration; Nunes-Nesi et al. 2005) and Arabidopsis (decreased photorespiration; Tomaz et al. 2010). The up-regulation of the photorespiratory pathway could be a general effect of TCA dysfunction in tomato cells because its increased activity was also observed in plants with impaired CS (Sienkiewicz-Porzucek et al. 2008), aconitase (Carrari et al. 2003) and mtIDH activity (SienkiewiczPorzucek et al. 2010). Impairment of the TCA cycle also leads to up-regulation of proteolysis and an increase in AA catabolism, providing an alternative source of organic acids and/or electrons used directly by mtETC. The increase in AA catabolism was observed in plants with impaired CS (Sienkiewicz-Porzucek et al. 2008), mitochondrial NADIDH (Sienkiewicz-Porzucek et al. 2010), succinyl CoA ligase (SCoAL; Studart-Guimarães et al. 2007) and Fum (Nunes-Nesi et al. 2007). The dysfunction of the matrix-localized AA catabolic pathway has a minor effect on growth and leaf N metabolism under non-stressed conditions. It was found that mutations in gene that encode ProDH or P5CDH have no impact on Pro concentration (Mani et al. 2002; Deuschle et al. 2004). The impairment activity of isovaleryl-CoA dehydrogenase also does not result in any significant changes in AA profile under non-stressed conditions (Araújo et al. 2010).
THE CHANGES OF N METABOLISM RESULTING FROM THE DYSFUNCTION OF mtETC The dysfunction of mitochondrial complex I is often associated with changes in the cellular redox state (Juszczuk, Szal & Rychter 2011 and references therein). For example, the consequence of complex I impairment is an increase in the cytosolic NADH/NAD ratio (Szal et al. 2008). Because the reduction of NO3- is limited mainly by the availability of reductants (Kaiser et al. 2000), reduction of NO3- could be promoted in mutants. Indeed, the accumulation of AAs in CMSII and css1 plants was observed (Dutilleul et al. 2005; © 2012 Blackwell Publishing Ltd, Plant, Cell and Environment
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Nakagawa & Sakurai 2006). In the best-characterized complex I mutant tobacco CMSII, an increase of asparagine (Asn) and Gln and a decrease of aspartic acid (Asp) and Glu was found (Dutilleul et al. 2005; Hager et al. 2010); however, these changes are not universal. Elevated Glu levels and unchanged Asn and Gln concentrations were found in the cucumber complex I mutant (Szal et al. 2010). In general, the dysfunction of complex I results in significant accumulation of arginine (Arg; Dutilleul et al. 2005; Nakagawa & Sakurai 2006; Szal et al. 2010), which is involved in the C/N signalling pathway in cells. In addition, the accumulation of Ala is a common response to complex I impairment (Dutilleul et al. 2005; Nakagawa & Sakurai 2006; Garmier et al. 2008; Szal et al. 2010). This might be the result of increased activity of Ala aminotransferase detoxifying cells from NH4+ as the concentration of NH4+ increases in complex I mutants (Dutilleul et al. 2005; Szal et al. 2010). This might be also because of the activation of the fermentative pathway leading to Ala accumulation, as is similarly observed when mtETC activity, is limited by low oxygen availability (Rocha et al. 2010). Moreover, accumulation of branched-chain AAs was found in plants with complex I dysfunction (Dutilleul et al. 2005; Meyer et al. 2009). Transcripts for a number of the components of the branched-chain AA degradation pathway were induced in ndufs4 plants (Meyer et al. 2009). Possibly, branched-chain AAs are alternative substrates for respiration as electrons from their degradation are transferred directly to ubiquinone (Ishizaki et al. 2005, 2006). Under non-stressed conditions, the changes in AOX expression do not influence primary N metabolism as was shown using aox1 mutants (Giraud et al. 2008; Vanlerberghe, Cvetkovska & Wang 2009); however, under stress conditions, the impact of decreased expression or overexpression of AOX on N metabolism indicates the essential role of this enzyme in the maintenance of a balanced C/N ratio in plant tissue (Sieger et al. 2005; Giraud et al. 2008; Watanabe et al. 2008). In the ucp1 mutant, a significant decrease in the flux of Gly to Ser was found (Sweetlove et al. 2006). Surprisingly, a reduced level of both Gly and Ser was observed in ucp1 plants, suggesting some kind of regulatory response to decreased flux through upstream steps of photorespiration (Sweetlove et al. 2006). The effect of ETFQO impairment on N metabolism under non-stress conditions is unclear because of the contradictory effects concerning Ala, Asp or Pro concentration that were observed in etfqo mutants (Ishizaki et al. 2005 versus Ishizaki et al. 2006).
MITOCHONDRIAL NO PRODUCTION NO is considered an important signalling molecule and is implicated in the regulation of multiple plant processes, including photomorphogenesis, leaf expansion, stomatal closure and response to various abiotic and biotic stresses (Beligni & Lamattina 2000; Neill et al. 2002). NO is also involved in controlling programmed cell death via S-nitrosylation of different proteins (Leitner et al. 2009).
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During the past few decades, among the well-documented paths of NO production (e.g. cytosolic NR), the possibility of NO generation in mitochondria due to NO synthase (NOS) activity or mtETC has been widely discussed. Different isoforms of NOS that catalyse the conversion of L-Arg to NO and citrulline are known in mammals. In plants, NOS activity has been linked to two protein types: the variant (not mitochondrial) form of the P protein of the GDC; (Chandok et al. 2003) and the mitochondrially localized AtNOS1 (Guo, Okamoto & Crawford 2003), later renamed At NO-associated 1 (AtNOA1; Crawford et al. 2006). Unfortunately, the results of both proteins were irreproducible. In particular, a lively discussion about the potential role (NOS or GTPase activity) and localization (mitochondrial or chloroplast) of AtNOA1 was conducted among several research groups (Guo & Crawford 2005; Crawford et al. 2006; Guo 2006; Zemojtel et al. 2006; Moreau et al. 2008). The involvement of mtETC in NO formation in plant cells has been confirmed by several data (Tischner, Planchet & Kaiser 2004; Planchet et al. 2005); however, debate remains regarding which part of mtETC may be the source of NO. In mtETC, NO2- may be used as an alternative electron acceptor (NO2- is reduced to NO); therefore, mitochondrial NO generation depends on cytosolic NR activity (supplying NO2-). The most plausible candidate responsible for mitochondrial NO emission is complex IV, but complex III, cytochrome c and AOX have also been proposed (Gupta & Igamberdiev 2011). These components were suggested based on the observation that nitrite-dependent NO generation by mitochondria decreases in the presence of respiratory inhibitors including antimycin A or myxothiazol (complex III inhibitors; Modolo et al. 2005; Planchet et al. 2005) and salicylhydroxamic acid (AOX inhibitor; Planchet et al. 2005). Cvetkovska & Vanlerberghe (2012) recently showed that AOX is not directly responsible for NO production but rather modulates NO generation by controlling the rate at which electrons leak from mtETC to NO2-. The participation of other suggested sites of NO2- to NO reduction in mtETC has not yet been precisely quantified. Mitochondrial NO production, in addition to being involved in retrograde signalling (e.g. Aox1 expression is induced by NO; Huang, von Rad & Durner 2002), influences the functioning of mitochondrion per se. NO inhibits COX activity (Millar & Day 1996), and prolonged exposure of NO to mitochondria causes complex I dysfunction (Brown & Borutaite 2002). Besides, the activities of aconitase and GDC are modulated by NO (Palmieri et al. 2010; Gupta et al. 2012). The inhibition of aconitase by NO increases citrate concentration and thereby improves the availability of C skeletons originating in the mitochondria for AA synthesis of the GS-GOGAT cycle (Gupta et al. 2012). The modulation of GDC activity by NO-dependent S-nitrosylation influences the redox state of mitochondria, but the role of this regulation has not yet been precisely identified (Palmieri et al. 2010). Another question concerns the conditions and plant organs in which NO may be produced in mitochondria. It
has been suggested that mitochondrial nitrite reduction occurs only when the oxygen concentration in a tissue is very low (Gupta et al. 2011), a condition that might occur during flooding stress. Secondly, it was demonstrated that mitochondria show nitrite-dependent NO emission in the roots but not the leaves of higher plants (Gupta, Stoimenova & Kaiser 2005). Both the above dogmas were recently disproven by Cvetkovska & Vanlerberghe (2012), who measured NO in tobacco leaf mitochondria under aerobic conditions. Most likely, under optimal conditions, NO is formed in quantities sufficient for signalling function, but it becomes an important metabolite playing a role in bioenergetics of the cell in anaerobic tissues (Stoimenova et al. 2007).
SUMMARY To sustain optimal growth and development of plants, cellular N metabolism must be closely coordinated with other metabolic pathways. The aim of our review was to emphasize the important role of mitochondria in this coordination. Because of their plasticity, plant mitochondria might not only provide energy and C skeletons, but might also sustain a balanced C/N ratio and redox reactions under stress conditions. We have shown that mitochondria are important players during N uptake, NO3- reduction, NH4+ assimilation and reassimilation processes occurring at subsequent stages of leaf development from young leaves until the late phase of senescence. Another aspect that should be considered is the participation of mitochondria in NO production.
ACKNOWLEDGMENTS We are grateful to Prof. A.M. Rychter (University of Warsaw) for critically reading the manuscript. This work was supported by Grant No. N303 401536 from the Ministry of Science and Higher Education, given to B.S.
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