Journal of Experimental Botany, Vol. 53, No. 370, pp. 905-916,
April 15, 2002
© 2002 Oxford University Press
Original Papers |
Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation
Institut de Biotechnologie des Plantes, CNRS UMR8618, Université Paris Sud-XI, 91405 Orsay Cedex, France
Received 18 July 2001; Accepted 8 October 2001
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Abstract |
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 Top Abstract Ammonium production and the...Isocitrate dehydrogenases and...Transamination reactions can...The mobility of 2-oxoglutarate...Plant cell 2-oxoglutarate levels...Regulation by 2-oxoglutarate...Conclusions and perspectivesReferences |
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Ammonium is the reduced nitrogen form available to plants for assimilation into amino acids. This is achieved by the GS/GOGAT pathway that requires carbon skeletons in the form of 2-oxoglutarate. To date, the exact enzymatic origin of this organic acid for plant ammonium assimilation is unknown. Isocitrate dehydrogenases and aspartate aminotransferases have been proposed to carry out this function. Since different (iso)forms located in several subcellular compartments are present within a plant cell, recent efforts have concentrated on evaluating the involvement of these enzymes in ammonium assimilation. Furthermore, several observations indicate that 2-oxoglutarate is a good candidate as a metabolic signal to regulate the co-ordination of C and N metabolism. This will be discussed with respect to recent advances in bacterial signalling processes involving a 2-oxoglutarate binding protein called PII.
Key words: Ammonium assimilation, aspartate aminotransferase, isocitrate dehydrogenase, 2-oxoglutarate, PII protein.
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Ammonium production and the GS/GOGAT cycle |
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TopAbstractAmmonium production and the...Isocitrate dehydrogenases and...Transamination reactions can...The mobility of 2-oxoglutarate...Plant cell 2-oxoglutarate levels...Regulation by 2-oxoglutarate...Conclusions and perspectivesReferences |
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Inorganic nitrogen assimilation, in the form of ammonium, onto carbon skeletons for the production of amino acids is one of the most important biochemical processes in plants. Although nitrogen is present in the atmosphere, it is often a limiting nutrient for plant growth as only some plants are capable of using this N source. In such plants, especially the leguminous plants, atmospheric dinitrogen is converted to ammonium by a nitrogenase activity present in rhizobia bacteria that establish a symbiotic interaction via specialized root organs called nodules. This ammonium is rapidly assimilated into nitrogenous compounds for export out of the nodules. Most higher plants obtain nitrogen from soil nitrate, and to a lesser extent ammonium, arising from mineralization of organic matter or fertilizers. Since ammonium is the reduced N form used by plants for assimilation into amino acids and protein, nitrate is converted to ammonium by the sequential action of the cytosolic nitrate reductase (NR) and the chloroplastic nitrite reductase (NiR) (Lea and Ireland, 1999
). In addition to this influx of nitrogen, there is an important recycling of nitrogenous compounds within the plant that produces large amounts of ammonium that need to be reassimilated. A major source of ammonium, especially in C3 plants, arises in the mitochondria from the decarboxylation of glycine to serine by the glycine decarboxylase (GDC) as part of the photorespiratory cycle. Ammonium is also produced during the breakdown of storage proteins and the deamination of proteins and the catabolism of nucleic acids associated with senescence. Ammonium can be toxic to plant function and so it must be rapidly assimilated into non-toxic organic compounds. This is achieved by the GS/GOGAT cycle or pathway that is comprised of the two enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT) (Ireland and Lea, 1999). This pathway is of crucial importance since the glutamine and glutamate produced are donors for the biosynthesis of major N-containing compounds, including amino acids, nucleotides, chlorophylls, polyamines, and alkaloids (Lea and Ireland, 1999). For the GS/GOGAT cycle to work, there is a strict requirement for N metabolism to interact with C metabolism since GS activity requires energy in the form of ATP and the GOGAT uses C-skeletons and reductant in the form of 2-oxoglutarate and reduced ferredoxin or NADH, respectively. In higher plant leaves, a specific GS isoform (named GS2) and a ferredoxin-dependent GOGAT (Fd-GOGAT), located in mesophyll cell chloroplasts, are responsible for the majority of plant ammonium assimilation (Fig. 1). The GS/GOGAT pathway transfers ammonium to glutamate to form glutamine, then the fixed N is transferred to 2-oxoglutarate thus leading to the biosynthesis of two molecules of glutamate. The key organic acid, 2-oxoglutarate, is mainly derived from sugar respiration and amino acid transamination reactions. However, the exact enzymatic origin of this C-compound for plant ammonium assimilation is still unknown since a variety of 2-oxoglutarate-synthesizing enzymes and isoenzymes exist in several subcellular compartments within the same plant cell. In the literature, the main candidates are isocitrate dehydrogenases (Gálvez et al., 1999) and aspartate aminotransferases (AspAT) (Lam et al., 1996). Whether these enzymes play overlapping (redundant) or distinct (non-redundant) roles is an important question. Recent data, often involving molecular genetics and plant engineering, have shed some light on this problem and argue in favour of specific non-redundant roles for these different enzymes (Lancien et al., 2000). Furthermore, over the years, it has become evident that certain signalling molecules like hormones (Sakakibara et al., 1999) and key C and N metabolites play an important role in co-ordinating plant C and N metabolisms (Coruzzi and Bush, 2001). Since 2-oxoglutarate levels can reflect cell C/N status, this metabolite could be involved in the monitoring and signalling of C/N balance to the plant regulatory machinery and emerging evidence for such a role will be discussed.
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Special attention will now be paid to recent advances concerning isocitrate dehydrogenases and aspartate aminotransferases and their role in amino acid synthesis. A major difference between these two enzymatic origins of 2-oxoglutarate for GS/GOGAT functioning is that an isocitrate dehydrogenase allows for net glutamate synthesis while an AspAT origin leads to the synthesis of aspartate (Fig. 1
).
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Isocitrate dehydrogenases and net glutamate synthesis |
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TopAbstractAmmonium production and the...Isocitrate dehydrogenases and...Transamination reactions can...The mobility of 2-oxoglutarate...Plant cell 2-oxoglutarate levels...Regulation by 2-oxoglutarate...Conclusions and perspectivesReferences |
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Isocitrate dehydrogenases produce 2-oxoglutarate from isocitrate and require a pyridine nucleotide as cofactor. Plants have two types of isocitrate dehydrogenase that use different cofactors; one is dependent upon NAD (IDH) while the other uses NADP (ICDH). IDH is a tricarboxylic acid (TCA or Krebs) cycle enzyme, located uniquely in the mitochondria of eukaryotic cells. Distinct ICDH isoenzymes are located within the cytosol (Gálvez et al., 1996
), mitochondria (Gálvez et al., 1998), plastids (Gálvez et al., 1994) and peroxisomes (Corpas et al., 1999) and their precise physiological function(s) are still unknown. Apart from their possible role in amino acid synthesis, it is probable that these isoenzymes are important for NADPH production. In yeast, the peroxisomal ICDH has been shown to be essential in the production of NADPH required for the ß-oxidation of unsaturated fatty acids (van Roermund et al., 1998). More recently, in mice cells, it has been shown that the mitochondrial ICDH protects mitochondria against oxidative stress due to the production of NADPH needed for glutathione reduction (Jo et al., 2001).
Two hypotheses involving different isocitrate dehydrogenases and subcellular compartments are described for GS/GOGAT functioning. It has been proposed that mitochondria are the source of the 2-oxoglutarate, produced by the IDH, this 2-oxoglutarate would leave the Krebs cycle and the mitochondria to be imported into the plastids (Fig. 2). The second hypothesis is that cytosolic ICDH could synthesize the 2-oxoglutarate needed for amino acid synthesis. In this pathway, citrate transported from the mitochondria to the cytosol is used by the cytosolic aconitase to generate the isocitrate required for 2-oxoglutarate synthesis by the cytosolic ICDH. This 2-oxoglutarate, of cytosolic origin, is then imported into the plastids (Fig. 2).
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Does cytosolic NADP-dependent isocitrate dehydrogenase synthesize the 2-oxoglutarate required for GOGAT functioning?
Although an ICDH is found in the plastid, the site of the GS/GOGAT pathway, it was proposed that cytosolic ICDH could play a major role in 2-oxoglutarate production for amino acid synthesis (Chen and Gadal, 1990
). To date, there is no direct evidence supporting this idea but observations are abundant in the literature that could argue in its favour.
The cytosolic isoform is responsible for 95% of the total ICDH activity measured in green tobacco leaf extracts (Gálvez et al., 1994). It has been shown to be the predominant isoform in tomato fruit (Gallardo et al., 1995) and potato (Kruse et al., 1998), and the only detectable ICDH activity in pine cotyledons (Palomo et al., 1998). A comparison of isoenzyme patterns from 15 species showed that cytosolic ICDH activity was always predominant in leaf extracts (Chen, 1998). Such observations consolidate the idea that the cytosol is a major site for 2-oxoglutarate production. All purified ICDH isoenzymes are homodimers composed of a subunit of approximately 47 kDa and they each show similar low substrate Km values (Km isocitrate 11–80 µM, Km NADP 4–16 µM, for more details see Gálvez et al., 1999). Again, this has been taken as an argument for the ICDH origin. Protein sequence data suggest that ICDH isoforms are closely related and have been highly conserved during evolution. An analysis of ICDH sequences from eukaryotes indicated that the cytosolic and mitochondrial isoforms arose from independent gene duplications in animals and fungi (Nekrutenku et al., 1998). This is probably the case in the plant kingdom and could explain their similar kinetic properties. Surprisingly, the Arabidopsis thaliana genome appears to have only three ICDH genes (Table 1). Each ICDH gene shows a conserved 15 exon/14 intron structure. Chromosome I contains the genes that would encode the cytosolic and peroxisomal (based on the presence of a small C-terminal extension carrying the tripeptide SRL; Hayashi et al., 1997) enzymes that appear to be closely related. The third gene, located on chromosome V, could encode both the mitochondrial and chloroplastic isoforms perhaps due to a hypothetical common function in the production of NADPH in response to oxidative stress conditions. To date, there is no experimental evidence to support such an idea. Furthermore, transgenic tobacco transformed with a chimeric gene composed of the 35S CaMV promoter, the presequence of the equivalent tobacco cDNA and the coding sequence of a modified green fluorescent protein (GFP) led to GFP detection only in mitochondria (Gálvez et al., 1998).
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A number of observations have linked cytosolic ICDH with amino acid metabolism. Tobacco plants deficient in NR activity and showing an accumulation of nitrate and 2-oxoglutarate, but low glutamine levels, exhibited significant increases in cytosolic ICDH, GS2, Fd-GOGAT, phosphoenolpyruvate carboxylase (PEPc), and pyruvate kinase transcript levels (Scheible et al., 1997
). These changes were interpreted as a functional co-ordination of cytosolic ICDH, GS2 and Fd-GOGAT, thus suggesting a role for cytosolic ICDH in organic acid production for nitrate assimilation (Stitt, 1999). In pine, a correlation was found, at the transcript level, between ICDH and GS during seed germination and between ICDH, GS and Fd-GOGAT during chloroplast biogenesis. However, this correlation was not observed during the advanced stages of cotyledon development (Palomo et al., 1998). The authors suggested that pine ICDH has several roles, including GS-GOGAT functioning. A similar conclusion was reached from cytosolic ICDH expression studies during potato plant development and with detached potato leaves. The changes in cytosolic ICDH transcript and activity levels brought about by light and mimicked by nitrate and sucrose, as seen for other N metabolism genes (Stitt, 1999), were indicative of a role in primary N metabolism (Fieuw et al., 1995). Similarly, induced senescence of detached leaves led to an increase in ICDH activity and while leaf total protein levels decreased, ICDH protein was stable (Fieuw et al., 1995). Leaf cytosolic ICDH transcript levels were also found to be stable throughout the development of tobacco plants, even in ageing leaves where mRNA levels for other enzymes diminished (Masclaux et al., 2000). These observations suggest a role for cytosolic ICDH in the cycling, redistribution and export of amino acids during senescence. A similar role was proposed following a comparison of ICDH activity and properties during the ripening of tomato fruits in which a 2–3-fold increase in cytosolic ICDH activity was found during glutamate accumulation (Gallardo et al., 1995).
However, several observations argue against a role for cytosolic ICDH in the production of 2-oxoglutarate for ammonium assimilation. The preferential localization of cytosolic ICDH in leaf (Gálvez et al., 1996; Fieuw et al., 1995) and root vascular tissues (Boiffin et al., 1998) is not in agreement with its proposed role in the major ammonium assimilatory pathway. Interestingly, vascular tissues contain a cytosolic GS and an NADH-GOGAT (Ireland and Lea, 1999), therefore cytosolic ICDH could synthesize 2-oxoglutarate for other plant metabolic functions involving glutamine and/or glutamate production. This could explain the increase in ICDH transcript and activity levels in senescing potato leaves (Fieuw et al., 1995), thus reflecting a role in amino acid remobilization and export.
When wild-type tobacco plants were submitted to a short-term N-starvation followed by nitrate or ammonium re-supply, cytosolic ICDH mRNA levels were not significantly affected (Lancien et al., 1999). This apparent contradictory observation when compared to the tobacco NR mutant studies (Scheible et al., 1997) probably reflects the differences in the experimental conditions and plant history. In one case (Scheible et al., 1997), nitrate assimilation was severely reduced or absent while in the second (Lancien et al., 1999), nitrate assimilation was active. The generation of different metabolic signals arising from a different C/N status could have affected plant response. Since 2-oxoglutarate is at the interface of C and N metabolism, it is possible that its site and level of production could be modified according to specific physiological conditions and metabolic fluxes.
A major argument against the role of cytosolic ICDH in the major ammonium assimilatory pathway comes from the analysis of transgenic plants in which cytosolic ICDH activity was significantly reduced. In potato (Kruse et al., 1998) and tobacco (M Hodges, unpublished data) retaining only 8% and 5% of their total ICDH activity, respectively, no deleterious phenotype affecting growth or flowering was detected. A detailed analysis of the potato plants showed no noticeable modification in C or N metabolism and amino acid levels were not significantly altered (Kruse et al., 1998). Therefore, it appears that cytosolic ICDH is not essential for ‘normal’ GS/GOGAT cycle functioning. However, the residual cytosolic ICDH activity might be sufficient to allow for ammonium assimilation or, alternatively, compensatory pathways giving rise to metabolic plasticity could have been induced in such transgenic lines.
Mitochondrial NAD-dependent isocitrate dehydrogenase could synthesize the 2-oxoglutarate required for GOGAT functioning
According to Miflin and Lea, mitochondria are the source of 2-oxoglutarate for ammonium assimilation, produced by an IDH (Miflin and Lea, 1980). To date, this role for IDH remains an hypothesis. Indeed, IDH function is still poorly understood in plants, especially in N-assimilating tissues such as leaves.
IDH is announced as a key Krebs cycle enzyme. To date, the best-characterized IDH is from yeast where two genes encode different subunits called IDH1 and IDH2 (Keys and McAlister-Henn, 1990; Table 2). Yeast IDH is an octomer composed of a tetramer of IDH1–IDH2 dimers (Panisko and McAlister-Henn, 2001). Initially it was proposed that IDH2 was responsible for the catalytic activity and IDH1 had a regulatory role, although recently it has been shown that certain residues located on IDH1 are required to form the active catalytic site (Panisko and McAlister-Henn, 2001). Its essential role in the Krebs cycle has been shown from the acetate- phenotype of different IDH yeast mutants. Interestingly, these mutants were not glutamate auxotrophs due to the presence of the cytosolic and mitochondrial ICDH enzymes (Zhao and McAlister-Henn, 1996). In mammals, IDH is also heteromeric; three genes exist that encode the so-called 
, ß and 
subunits (Table 2). Protein sequence comparisons and biochemical studies using recombinant protein technology have shown that the subunit is equivalent to IDH2, being essential for catalytic activity (Kim et al., 1999). The IDH complex in vivo is a tetramer composed of 2 subunits, a ß and a subunit, although recombinant IDH made of and ß or and subunits do show an activity but this is 10-fold lower than the tetrameric IDH complex composed of all three subunits (Kim et al., 1999).
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In plants, both a low measurable in vitro activity and high instability have hampered progress in an understanding of plant IDH function and regulation. Only recently, has an IDH been purified to homogeneity from a plant source, the unicellular green algae, Chlamydomonas reinhardtii (Martínez-Rivas and Vega, 1998
). It was found to be an octomer composed of 45 kDa subunits. This IDH was inhibited by NADH (competitive with respect to NAD) and NADPH (non-competitive with respect to NAD), but there was no observed activator effect of either AMP (as in yeast) or ADP (as in mammals). Two biochemically distinct forms of IDH have been partially purified and characterized from etiolated pea plants (McIntosh, 1997). An IDH activity was found in the soluble mitochondrial matrix, while a second activity was associated with the mitochondrial membrane fraction. These two putative IDH forms had similar enzymatic properties. It was proposed that the membrane-bound IDH form could be associated with an integral membrane protein, such as a component of respiratory chain complex I, a transhydrogenase or a dicarboxylate transporter (McIntosh, 1997). The Arabidopsis genome contains five putative IDH genes (Table 1), although only four of them have their corresponding expressed sequence tags (ESTs). Two of the genes have a conserved 7 exon/6 intron structure, they are located on chromosomes III and V, and encode proteins that resemble yeast IDH2 (the ‘catalytic’ subunit). The three remaining genes have a conserved 4 exon/3 intron structure, they are located on chromosomes II and IV and encode proteins that are similar to yeast IDH1. cDNAs corresponding to the IDH1 and IDH2 genes have been described previously (Behal and Oliver, 1998). In tobacco, three different cDNAs that encode different IDH subunits, named IDHa, IDHb and IDHc, have been isolated and characterized (Lancien et al., 1998; Table 2). They each have a predicted MM of approximately 36 kDa after removal of the mitochondrial presequence. IDHa was found to be necessary for enzymatic activity while IDHb and IDHc were given regulatory functions. These conclusions are based on the functional complementation of yeast IDH mutants by the tobacco IDH cDNAs. It was possible to complement the 
IDH1 mutant with either IDHb or IDHc and only the association of IDHa with either IDHb or IDHc created a functional enzyme in double mutant yeast strains lacking IDH function (Lancien et al., 1998). Although, an IDH activity was not detected in the mitochondria isolated from the complemented IDH1/IDH2 yeast strains, an IDH activity has since been measured from extracts of E. coli expressing the same tobacco IDH subunit combinations (T Lemaitre and M Hodges, unpublished data). Such observations indicate that plant IDH is heteromeric and raise the question of whether IDH is present in several different forms in higher plants, perhaps with specific functions.
Recently, data has been presented that suggests a role for IDH in N assimilation. IDH transcript levels were found to increase when N-starved tobacco plants were resupplied with either nitrate or ammonium (Lancien et al., 1999). Furthermore, a co-ordinated expression of IDH with other genes of the Krebs cycle (citrate synthase and aconitase), nitrate reduction (NR) and ammonium assimilation (GS) in N-starved tobacco plants after nitrate resupply was described in this work. Since the response kinetics were correlated to the nitrate assimilatory capacity of each organ, it was suggested to be signalled either by nitrate status or metabolites associated with its metabolism (since the changes were correlated to measurable NR activity).
However, a number of observations argue against this mitochondrial origin. IDH shows an extremely low in vitro measurable activity, relatively high substrate Km values when compared to ICDH (Km isocitrate 280–846 µM, Km NAD 150–800 µM) and an inhibition by NADH (Ki 70–420 µM) and NADPH (Ki 300–450 µM) (see Gálvez et al., 1999, for more details). Of course, the in vivo IDH activity might be potentially higher than that measured in vitro, since it could be regulated and/or depend upon certain undefined physiological conditions. For instance, IDH might be associated with other Krebs cycle enzymes in a structured enzymatic complex termed a metabolon (Vélot et al., 1997). This could enhance in vivo organic acid flux channeling and/or protect IDH (and other TCA cycle dehydrogenases) against inhibition by the predicted high NADH levels present in the light. However, it is probable that NADH does not accumulate in mitochondria in the light. Reducing equivalents are believed to be rapidly and efficiently exported from the mitochondria to the cytosol by the action of the malate/oxaloacetate shuttle, driven by the activity of a yet to be isolated dicarboxylate transporter (Hanning et al., 1999). Furthermore, an alternative oxidase, stimulated by the accumulation of organic acids and reducing power, can bring about the oxidation of reduced NADH without ATP synthesis (Rhoads et al., 1998). In photosynthetic tissues, the presence of a functional Krebs cycle remains an unanswered question. In the light, the pyruvate dehydrogenase complex (PDC) is inhibited by a regulatory phosphorylation mechanism (Schuller et al., 1993) that is stimulated by ammonium. The important GDC activity, due to the photorespiratory pathway in leaf mitochondria, should inhibit all Krebs cycle dehydrogenases, including IDH, due to the production of NADH and, at the same time, inhibit the PDC due to the production of ammonium. However, photorespiration and C-skeleton production for ammonium assimilation should function simultaneously. Interestingly, in nitrate-grown sugar beet, photorespiratory ammonium was not immediately assimilated because of a C limitation arising from the low day-time respiratory activity (Schjoerring et al., 2000). The C-limitation was correlated to the measured 2-oxoglutarate levels; being lowest in the light and increasing 30-fold at the beginning of the dark period when respiration increased (Schjoerring et al., 2000). In these plants, ammonium accumulated in the apoplastic space (with some ammonia loss to the atmosphere) during the day and it was reassimilated during the night when respiration and GS activities both increased. However, this might be species-dependent since, in oil-seed rape, temperature-induced increases in photorespiratory rates did not lead to modified ammonium levels thus indicating that the ammonium was efficiently assimilated.
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Transamination reactions can produce 2-oxoglutarate, but do not lead directly to net Glu synthesis |
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TopAbstractAmmonium production and the...Isocitrate dehydrogenases and...Transamination reactions can...The mobility of 2-oxoglutarate...Plant cell 2-oxoglutarate levels...Regulation by 2-oxoglutarate...Conclusions and perspectivesReferences |
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In higher plants, aspartate aminotransferases are homodimeric enzymes composed of subunits of approximately 40 kDa that require pyridoxal phosphate as a cofactor. They can produce 2-oxoglutarate and aspartate via the reversible amino group transfer from glutamate to oxaloacetate (Figs 1, 3
). Their measured Km values are quite high, especially for glutamate (1–4 mM for aspartate, 4–36 mM for glutamate, 0.2–1 mM for 2-oxoglutarate, and 0.02–0.1 mM for oxaloacetate; see Lea and Ireland (1999) for more details). Several functions have been attributed to AspAT. These include a central role in C and N metabolism by the trapping and liberation of key oxo-acids that help co-ordinate N metabolism and amino acid synthesis with the availability of C skeletons from the Krebs cycle. When coupled to the GS/GOGAT cycle, an AspAT activity leads to a complete recycling of glutamate and the synthesis of aspartate. The crucial organic acid is no longer 2-oxoglutarate but oxaloacetate that can be produced in the cytosol by the activity of PEP carboxylase. The aspartate can be used as a precursor for the synthesis of other amino acids such as asparagine, threonine, isoleucine, leucine, valine, lysine, and methionine. Another important role for an AspAT is in the symbiotic N-assimilation of legumes. Although legume roots contain two distinct AspAT forms (AAT1 and AAT2), the (amylo)plastidial AAT2 form was shown to be responsible for the nodule-located activity (Vance et al., 1994). AspAT is also involved in the shuttling of redox equivalents between subcellular (the malate–aspartate shuttle in C3 plants) and cellular compartments (the C-shuttle from the mesophyll to the bundle sheath cells in C4 plants) (Lea and Ireland, 1999).
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Biochemical studies have shown the presence of distinct AspAT isoenzymes localized within plastids, cytosol, mitochondria, peroxisomes, and glyoxisomes (Lam et al., 1996
; Ireland and Lea, 1999). Five cDNAs (named Asp1 to Asp5; Table 1) have been isolated and characterized from Arabidopsis thaliana (Schultz and Coruzzi, 1995). The correspondence between a given gene and an AspAT isoenzyme has been proposed by using several approaches including activity gel analyses, the characteristics of putative N-terminal presequences (Schultz and Coruzzi, 1995) and the in vivo uptake of recombinant AspAT into isolated chloroplasts (Wilkie et al., 1995) (Table 1). Northern analyses have shown that each gene is expressed in the leaves, roots, flowers, and cotyledons of Arabidopsis; except Asp4 that could not be detected by this method. The cytosolic (Asp2) and chloroplastic (Asp5) isoforms appear to be most predominant in this model plant. Asp2 RNA levels were highly expressed in the roots (Schultz and Coruzzi, 1995). Specific, non-overlapping functions have been proposed for the different isoenzymes due to differences in their expression patterns and response to C and N compounds. For instance, in millet, N availability led to a positive effect on the mRNA levels of the cytosolic and mitochondrial enzymes but not those of the chloroplastic isoform (Taniguchi et al., 1995). Recently, an Arabidopsis thaliana asp2 mutant (generated from EMS-mutagenized Arabidopsis seeds), having 3–6% of the wild-type activity as determined by native gel assays, has been characterized. The plants showed a retarded growth phenotype. Furthermore, it was found that this cytosolic isoform was responsible for the synthesis of an aspartate pool during the light that was converted to asparagine in the dark for N-transport within the plant (Schultz et al., 1998; Fig. 3). In the model proposed by Coruzzi and co-workers, the cytosolic AspAT would furnish the GS/GOGAT cycle with 2-oxoglutarate (Schultz et al., 1998). These observations clearly show that this enzyme is functionally distinct from the other AspAT isoforms. It was proposed that the different organelle-localized isoforms might be involved mainly in redox shuttling. Interestingly, the absence of a phenotype in an asp5 mutant and the absence of a photorespiratory mutant phenotype in the asp2 mutant plants could indicate that both isoforms do not play a predominant role in the production of 2-oxoglutarate for the major ammonium assimilatory pathway.
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The mobility of 2-oxoglutarate between subcellular compartments is required for ammonium assimilation |
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TopAbstractAmmonium production and the...Isocitrate dehydrogenases and...Transamination reactions can...The mobility of 2-oxoglutarate...Plant cell 2-oxoglutarate levels...Regulation by 2-oxoglutarate...Conclusions and perspectivesReferences |
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It can be seen that 2-oxoglutarate can be synthesized in a number of different subcellular compartments, therefore its mobility could be of major importance in metabolic functioning (Fig. 2
). It remains possible that a limiting step in ammonium assimilation is 2-oxoglutarate transport rather than its rate of production. 2-oxoglutarate transport activities have been measured in both mitochondria and chloroplasts. Several cDNAs encoding a millet mitochondrial malate transporter have been isolated that, by protein sequence comparisons, resembles the 2-oxoglutarate transporter of mammals. It is not known whether the different cDNAs encode isoforms with specific functions (Taniguchi and Sugiyama, 1996). Interestingly, the Arabidopsis genome contains a single gene that encodes a protein that resembles the millet malate transporter (Table 1). In the case of mitochondrial 2-oxoglutarate production for ammonium assimilation, the recombinant millet malate transporter was capable of carrying out an in vitro 2-oxoglutarate/malate exchange (Taniguchi and Sugiyama, 1996). This could reflect the need to attain the obligatory replenishment of Krebs cycle intermediates if 2-oxoglutarate is removed from the mitochondria for glutamate synthesis. To date, it is not known whether mitochondrial dicarboxylate and tricarboxylate exchange occurs via the same transporter. However, the millet malate transporter (Taniguchi and Sugiyama, 1996) and the Arabidopsis homologue (N Picault, L Palmieri and M Hodges, unpublished data) can transport both citrate and 2-oxoglutarate.
The 2-oxoglutarate transporters of mitochondria and chloroplasts (2OGMT) are not isoforms, since they are structurally distinct. The mitochondrial transporter is a homo-dimeric protein composed of a 32 kDa subunit containing six transmembrane -helices that belongs to a super family of mitochondrial transporter proteins that include uncoupling proteins, dicarboxylate (DIC) transporters, 2-oxoglutarate transporters, and citrate transporters (Laloi, 1999). On the other hand, the chloroplastic 2OGMT is a single polypeptide showing 12 membrane-spanning regions (Weber et al., 1995). Although, plastids contain an ICDH and an AspAT, their activities are probably too low to sustain the required ammonium assimilatory capacity of this organelle. In planta, chloroplastic 2OGMT activity is coupled to that of a glutamate/malate translocator (Weber et al., 1995). In this double-translocator system, 2-oxoglutarate entry into the chloroplast is co-ordinated with glutamate export to the cytosol via malate recycling. The need to import this crucial organic acid into plastids is revealed in antisensed chloroplastic 2OGMT tobacco plants showing a reduced 2-oxoglutarate transport activity and a ‘photorespiratory mutant’ phenotype; chlorosis in air (high photorespiration) but normal growth in CO2-enriched air (low photorespiration) (A Weber, personal communication). This is explained by a limitation in the 2-oxoglutarate/glutamate exchange required for Fd-GOGAT activity and peroxisomal photorespiratory glycine synthesis.
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Plant cell 2-oxoglutarate levels fluctuate in response to C and N metabolism |
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TopAbstractAmmonium production and the...Isocitrate dehydrogenases and...Transamination reactions can...The mobility of 2-oxoglutarate...Plant cell 2-oxoglutarate levels...Regulation by 2-oxoglutarate...Conclusions and perspectivesReferences |
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In a plant leaf cell, 2-oxoglutarate is present at relatively low levels when compared to other organic acids (10-fold less than citrate and 30-fold less than malate; for example, Morcuende et al., 1998
). It has been shown that 2-oxoglutarate levels depend upon metabolic fluxes related to C- and N- metabolism, although it is not known whether 2-oxoglutarate fluctuations reflect a change in a given enzymatic activity. Indeed, 2-oxoglutarate levels are dependent on inorganic and organic N content and their related metabolism as well as nitrate and sugar levels. This has been studied in nitrate-accumulating NR-deficient tobacco that led to an increase in 2-oxoglutarate content under conditions where glutamine levels stayed low, starch accumulated and sucrose allocation was altered (Scheible et al., 1997). In wild-type tobacco plants, leaf 2-oxoglutarate levels responded to N starvation when glutamine levels were low, nitrate was absent, NR activity was reduced, and sucrose root allocation had increased (Lancien et al., 1999). Sucrose provided to detached tobacco leaves induced a dramatic rapid increase in 2-oxoglutarate levels without any subsequent change in NR activity (Morcuende et al., 1998). Taken together, such data indicate that plant cell 2-oxoglutarate levels can reflect C/N status, and therefore this organic acid could play a signalling role in the co-ordination of C and N metabolism. This is well established in bacterial systems. |
Regulation by 2-oxoglutarate signalling in bacteria and plants |
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TopAbstractAmmonium production and the...Isocitrate dehydrogenases and...Transamination reactions can...The mobility of 2-oxoglutarate...Plant cell 2-oxoglutarate levels...Regulation by 2-oxoglutarate...Conclusions and perspectivesReferences |
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In bacteria, the GlnB gene encodes a 2-oxoglutarate receptor protein, named PII, that is part of a cascade regulating GS activity at both the transcriptional and post-translational levels (Ninfa and Atkinson, 2000
). In E. coli, these two processes are linked to a signalling process monitoring C and N supply in which 2-oxoglutarate (indicating C sufficiency) and Gln (indicating N sufficiency) are the metabolic signals. PII interacts with NRII (a bifunctional kinase/phosphatase) that modifies the ability of NRI (an enhancer-binding transcription factor) to regulate GlnA gene expression. Low levels of 2-oxoglutarate allow the formation of an NRII–PII complex that inactivates NRI by dephosphorylation. High 2-oxoglutarate concentrations inhibit NRII–PII formation and NRI is kept in an active, phosphorylated state. This regulatory mechanism is superimposed with the control by reversible adenylation of the GS protein. Inactive, adenylated GS is stimulated by PII and Gln while active, deadenylated GS is found in the presence of low 2-oxoglutarate levels and uridylated PII (Ninfa and Atkinson, 2000, and references within).
In cyanobacteria, a gene has been isolated that encodes a protein similar to the above-mentioned bacterial PII. By contrast to Enterobacteria, this PII protein is covalently modified by serine phosphorylation rather than by uridylation. In Synechococcus PCC 7942, the in vitro PII kinase activity has been shown to be dependent on 2-oxoglutarate and ATP (Tandeau de Marsac and Lee, 1999). In Synechocystis 6803, N deprivation led to a 10-fold transcriptional activation of the PII gene. Preliminary results indicated a correlation between PII status and GlnA activity (García-Domínguez and Florencio, 1997) although the cyanobacterial genome does not contain gene orthologues encoding NRI and NRII. Interestingly, the PII of Synechococcus sp PCC7942 is involved in the regulation of nitrate and nitrite uptake (Tandeau de Marsac and Lee, 1999). In the presence of NH4+, the ABC-type transporter involved in nitrate and nitrate uptake is inactivated. This process depends on the presence of PII and its phosphorylation state since a PII null mutant shows no NH4+ control of this transporter activity and the presence of a non-phosphorylatable PII leads to an absence of nitrate uptake (Lee et al., 2000). In this cyanobacteria, low 2-oxoglutarate levels give rise to a non-phosphorylated PII and the inhibition of N uptake, whereas in the presence of high 2-oxoglutarate concentrations, PII is phosphorylated and the nitrate/nitrite transporter is active. It is believed that this regulation reflects changes in the interaction of PII with a subunit of the transporter complex. The in vivo phosphorylation state of PII also reflects the C/N balance of the cell. In the presence of nitrate and high CO2 concentrations or during N starvation, PII is phosphorylated, while in the presence of NH4+ or low CO2, PII is non-phosphorylated (Tandeau de Marsac and Lee, 1999).
A gene (GLB1) encoding a homologue of the bacterial PII protein has been isolated from Arabidopsis thaliana (Hsieh et al., 1998). The identified PII protein was chloroplast-localized. GLB1 was expressed in all organs tested and GLB1 transcript levels responded to C and N metabolites as well as light. To test whether this regulation was related to plant PII function, transgenic tobacco constitutively overexpressing GLB1 were analysed. These transgenic plants appeared to be altered in C/N sensing, as judged by an in vivo assay; glutamine reversal of sucrose induced anthocyanin accumulation (Hsieh et al., 1998). It is not known whether this PII protein is involved in the regulation of GS transcription or post-transcriptional control in plants. Recently, an external 2-oxoglutarate supply was found to induce an increase of GS1 mRNA in Arabidopsis roots (Oliveira and Coruzzi, 1999). Furthermore, an antagonistic effect of added amino acids, such as glutamine, on sucrose-induced GS expression was observed. The authors suggested that the metabolic regulation of GS expression in plants was controlled by the relative abundance of C-skeletons versus amino acids. Further biochemical and molecular studies are necessary to confirm if 2-oxoglutarate is really an important signal in a pathway controlling gene expression and involving a PII sensing protein. It is possible that in plants, the chloroplastic PII could have a role in N transport regulation, similar to that found in cyanobacteria.
However, 2-oxoglutarate is a direct regulator of enzymes such as cytosolic pyruvate kinase and PEP carboxylase, mitochondrial citrate synthase, and alternative oxidase; each involved in sugar/organic acid flux and redox control between cytosol and mitochondria. In this way, it is possible that this organic acid acts as a regulatory metabolite co-ordinating mitochondrial, cytosolic and chloroplastic metabolism so as to adjust C-skeleton production or cellular redox regulation to amino acid synthesis.
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Conclusions and perspectives |
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TopAbstractAmmonium production and the...Isocitrate dehydrogenases and...Transamination reactions can...The mobility of 2-oxoglutarate...Plant cell 2-oxoglutarate levels...Regulation by 2-oxoglutarate...Conclusions and perspectivesReferences |
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This review includes recent arguments for and against the role of different enzymes involved in 2-oxoglutarate production and transport for ammonium assimilation. It can be seen that there is no clear answer since the situation is rendered complex due to the presence of isoenzyme families and the high degree of compartmentalization. This probably reflects plant metabolic flexibility so as to respond to changing environmental conditions. For instance, in the case of the origin of 2-oxoglutarate for ammonium assimilation, the contradictory observations often found in the literature could indicate that different enzymes contribute depending on plant tissue and specific physiological condition(s). The identification and characterization of null mutants for a given (iso)enzyme should help us to better understand the non-redundant roles of the numerous 2-oxoglutarate-producing enzymes. This approach has already led to significant advances when considering other N-assimilation enzymes like GS and GOGAT isoforms. However, the characterization of N-assimilation mutants often remain partial and further progress to unravel the dynamic, flexible nature of plant metabolism will be made only if an integrated, whole plant approach is developed.
Over the last few years, it has become clear that C and N metabolisms are regulated to bring about the co-ordination essential for plant growth and development. Although a number of C and N compounds have been shown to modify gene expression and certain enzymatic activities, interesting and fundamental questions remain to be answered. Which metabolites are important in controlling the co-ordination of C and N metabolisms? How do the signals lead to the regulation of gene and enzyme activities? The idea that C and N status is monitored by changing 2-oxoglutarate levels is of great interest, but the role of this organic acid as a signalling molecule remains to be proven. The discovery in Arabidopsis of GLB1 that encodes a putative 2-oxoglutarate sensing protein opens up the door to examine such an hypothesis. In the years to follow, significant progress awaits to be made in this area of metabolic sensing and signalling in plants.
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Acknowledgments |
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I am grateful to Dr Andreas Weber for sharing unpublished data. I would like to thank Professor Pierre Gadal for his support and Susana Gálvez for many valuable discussions. I would like to say a big thank you to Evelyne Bismuth, Muriel Lancien, Nathalie Picault, Thomas Lemaitre, and Luigi Palmieri.
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Notes |
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1 Fax: +33169153423. E-mail: hodges{at}ibp.u\|[hyphen]\|psud.fr
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References |
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