Download Respiration and nitrogen assimilation: targeting

Journal of Experimental Botany, Vol. 62, No. 4, pp. 1467–1482, 2011
doi:10.1093/jxb/erq453
REVIEW PAPER
Respiration and nitrogen assimilation: targeting
mitochondria-associated metabolism as a means to enhance
nitrogen use efficiency
Christine H. Foyer1, Graham Noctor2,* and Michael Hodges2
1
Centre for Plant Sciences, Faculty of Biology, University of Leeds, Leeds LS2 9JT, UK
Institut of Biologie des Plantes, UMR8618 CNRS/Université de Paris-Sud 11, Bâtiment 630, Université de Paris-Sud 11, F-91405
Orsay cedex, France
2
* To whom correspondence should be addressed: E-mail: [email protected]
Received 2 November 2010; Revised 10 December 2010; Accepted 15 December 2010
Abstract
Considerable advances in our understanding of the control of mitochondrial metabolism and its interactions with
nitrogen metabolism and associated carbon/nitrogen interactions have occurred in recent years, particularly
highlighting important roles in cellular redox homeostasis. The tricarboxylic acid (TCA) cycle is a central metabolic
hub for the interacting pathways of respiration, nitrogen assimilation, and photorespiration, with components that
show considerable flexibility in relation to adaptations to the different functions of mitochondria in photosynthetic
and non-photosynthetic cells. By comparison, the operation of the oxidative pentose phosphate pathway appears to
represent a significant limitation to nitrogen assimilation in non-photosynthetic tissues. Valuable new insights have
been gained concerning the roles of the different enzymes involved in the production of 2-oxoglutarate (2-OG) for
ammonia assimilation, yielding an improved understanding of the crucial role of cellular energy balance as a broker
of co-ordinate regulation. Taken together with new information on the mechanisms that co-ordinate the expression
of genes involved in organellar functions, including energy metabolism, and the potential for exploiting the existing
flexibility for NAD(P)H utilization in the respiratory electron transport chain to drive nitrogen assimilation, the
evidence that mitochondrial metabolism and machinery are potential novel targets for the enhancement of nitrogen
use efficiency (NUE) is explored.
Key words: Carbon–nitrogen interactions, isocitrate dehydrogenase, mitochondria, nitrate assimilation.
Introduction
Whilst the pathways of primary nitrogen assimilation, respiration, and the associated process of photorespiration are well
defined it is only since the application of reverse genetic
strategies that the high level of interaction between them has
begun to be fully appreciated (Bauwe et al., 2010; Sweetlove
et al., 2010). However, in terms of whole cell energy status the
exact contribution of respiration is dependent on cell type.
Moreover, fundamental questions remain, particularly concerning the degree of inhibition of the tricarboxylic acid
(TCA) cycle in the light. While TCA cycle flux is reduced in
leaves in the light when compared to that seen in the dark,
data obtained from enzyme measurements (Tovar-Mendez
et al., 2003) and isotope labelling profiles (Tcherkez et al.,
2005) are not easily reconciled with the results from transgenic
plants (Fernie et al., 2004). The operation of different flux
modes in the light has been suggested as a possible solution to
this problem (Sweetlove et al., 2010).
Whilst the co-ordination of the pathways of primary
nitrogen assimilation and respiration has been presumed for
decades, our understanding of the precise details of how this is
achieved remains incomplete. In tissues that lack photosynthesis such as roots, the reliance on mitochondrial oxidative
phosphorylation to meet the energy demands of the cell,
including nitrogen assimilation, greatly simplifies matters.
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1468 | Foyer et al.
However, even such general assumptions may be challenged
as it appears that some enzymes of the photorespiratory
pathway could be expressed in a root-specific fashion (NunesNesi et al., 2010). Moreover, while certain mitochondrial TCA
cycle reactions are required to support cytosolic/vacuolar
nitrate accumulation (Nunes-Nesi et al., 2007), a recent study
has suggested a major role of night-stored molecules in
providing 2-OG for glutamate synthesis in illuminated rape
seed (Gauthier et al., 2010). Some of these issues might be
resolved by genetic manipulation of individual enzymes of the
TCA cycle, as discussed below. Whilst our understanding of
related signal transduction cascades and associated retrograde
signalling from the mitochondria to the nucleus remains
fragmentary, there is increasing sophistication in the approaches that are being applied to gain a better resolution of
metabolism and its regulation (Niittylae et al., 2009).
Increasing evidence demonstrates the importance of
maintaining the cellular energy balance in the co-ordination
of primary nitrogen assimilation, respiration, and photorespiration. For example, an Arabidopsis thaliana mutant
deficient in the expression of the uncoupling protein
AtUCP1, which is an integral component of the inner
mitochondrial membrane, revealed a specific inhibition of
photorespiration (Sweetlove et al., 2006). Moreover, the ucp1
mutants displayed dramatically reduced rates of carbon
dioxide assimilation linked to a reduced rate of photorespiratory glycine oxidation (Sweetlove et al., 2006). Components of the mitochondrial electron transport chain such
as the non-phosphorylating bypass proteins (alternative
oxidases and internal NADH dehydrogenases) and the UCP
therefore appear to be essential for the proper maintenance
of intracellular redox gradients. In this context, uncoupling
proteins might be engaged when the demand for oxidation of
NADH is high, and so allow increased TCA cycle flux
(Smith et al., 2004). Moreover, engagement of the respiratory
alternative oxidase and the internal NADH dehydrogenases
would provide mechanisms whereby electron transport could
proceed without proton translocation and therefore could
fulfil similar roles to the UCP. The following consideration
of the complex interactions between respiration and nitrogen
metabolism will therefore focus on aspects of respiratory
metabolism and associated processes that generate biosynthetic precursors and also regulate the cellular energy balance
in order to ensure an efficient appropriate allocation of essential resources. The review considers two key related
aspects: first, current knowledge of the different enzymes that
contribute the key C skeleton, 2-oxoglutarate (2-OG) to
ammonia assimilation and, second, recent studies that have
analysed how genetic manipulation of the TCA cycle and
mitochondrial electron transport components impacts on N
metabolism and related processes.
Respiratory production of carbon skeletons
for amino acid synthesis
Ammonium can be produced by a number of metabolic
pathways and enzymatic reactions, such as the reduction of
nitrate to nitrite to ammonium via the action of nitrate and
nitrite reductases (NR, NiR), the action of glycine decarboxylase (GDC) during the photorespiratory cycle, and the
degradation of proteins during senescence, to name but
three. Irrespective of the ammonium-producing pathway, in
plant cells, it is (re)assimilated into organic molecules mainly
by the action of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle, although it could also possibly
be assimilated via glutamate dehydrogenase (GDH)
(Ireland and Lea, 1999). In the first step of the GS/GOGAT
cycle, ammonium is fixed to glutamate by GS and ATP is
consumed (Fig. 1). Then, GOGAT takes a molecule of
glutamine and makes two molecules of glutamate by
a reaction that requires carbon skeletons in the form of the
organic acid, 2-OG. This reaction also consumes a reductant, either reduced ferredoxin (Fd) via photosynthetic
electron transfer or NADH depending on the form of
GOGAT (Fd or NADH-GOGAT). In the case of GDH,
ammonium is directly fixed to 2-OG to form glutamate and
either NADH or NADPH are oxidized. However, GDH is
not a major ammonium-assimilating route in plant cells but
a stress/senescence-associated enzyme involved in the production of 2-OG from glutamate. By contrast, net ammonia
assimilation requires a source of 2-OG. Current understanding suggests that this 2-OG is generated through the
partial respiration of sugars or sugar-phosphates in which
the final step is catalysed by isocitrate dehydrogenases
(IDH). This section of the review discusses this small family
of 2-OG-producing enzymes and their roles in N assimilation and in redox-stress related NADPH production.
IDH produces 2-OG and liberates CO2 by the oxidative
decarboxylation of isocitrate (Fig. 1). This requires a pyridine
nucleotide co-factor (NAD or NADP) and a divalent cation
(e.g. magnesium or manganese). Plants contain two types of
IDH that use different co-factors. One is NADP-dependent
(NADP-IDH), while the other is NAD-dependent (NADIDH). Therefore, IDH activity also generates either NADPH
or NADH. The NAD-IDH is located uniquely in the
mitochondria of eukaryotic cells where it is part of the TCA
cycle. On the other hand NADP-IDH is found in several cell
compartments. Work in several species has established that
these are the cytosol, plastids, mitochondria, and peroxisomes (Gálvez et al., 1994, 1996, 1998; Corpas et al., 1999).
Their exact physiological function(s) are still under debate
(see Gálvez et al., 1999; Lancien et al., 2000; Hodges, 2002;
Hodges et al., 2003, for reviews).
Current knowledge of Arabidopsis genes encoding IDH is
summarized in Fig. 2. NAD-IDH is heteromeric and each
subunit (of approximately 35-37 kDa) is encoded by a
different gene. In Arabidopsis thaliana, five NAD-IDH genes
are expressed and by homology with the well-characterized
yeast enzyme, where IDH1 encodes a regulatory subunit and
the catalytic subunit is encoded by IDH2 (Keys and
McAlister-Henn, 1990), Arabidopsis contains two genes for
‘catalytic’ and three genes for ‘regulatory’ subunits. In
Arabidopsis rosettes, four of these genes (At4g35260,
At2g17130, At5g03290, and At3g09810) are expressed while
the fifth gene (At4g35650) is expressed mainly in pollen
Respiration and nitrogen metabolism | 1469
Fig. 1. Schematic overview of the compartmentation of some of the interactions between carbon and nitrogen during ammonia
assimilation. The emphasis is placed on reactions discussed in this review. For simplicity, metabolism of 2-OG through the TCA cycle is
not shown, while redox cofactors are shown only for nitrate reduction, ammonia assimilation, IDH, and the mitochondrial electron
transport chain. Dotted lines indicate movement, transport or a condensed series of several reaction steps. Although not shown, some
reactions are reversible (e.g. interconversion of OAA and malate; see Fig. 3). Chloroplast and mitochondrial transporters shown are
based on the studies of Weber et al. (1995), Picault et al. (2002), Renné et al. (2003), Sugiura et al. (2007), and Palmieri et al. (2008).
Numbered reactions are catalysed by (1) glycolytic enzymes; (2) phosphoenolpyruvate carboxylase; (3) pyruvate kinase; (4) malate
dehydrogenase; (5) malic enzyme; (6) pyruvate dehydrogenase; (7) citrate synthase; (8) aconitase. Abbreviations: AcCoA, acetylcoenzyme A; AltDH, mitochondrial alternative dehydrogenases; AOX, mitochondrial alternative oxidase; CI, CII, CIII, CIV, complexes I, II,
III, and IV of the mitochondrial electron transport chain; Cbf, cytochrome b6f complex; Cit, citrate; Cyt c, cytochrome c; DiC,
mitochondrial inner membrane dicarboxylate carrier; DiT1, DiT2, chloroplast inner envelope dicarboxylate exchangers; DTC,
mitochondrial inner membrane dicarboxylate/tricarboxylate carrier; Fd, ferredoxin; GS, glutamine synthetase; GOGAT, glutamate
synthase; IDH, isocitrate dehydrogenase; Isocit, isocitrate; Mal, malate; NiR, nitrite reductase; NiT, nitrite transporter; NR, nitrate
reductase; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; PSI, PSII, photosystems I and II; Pyr, pyruvate; PyrT, pyruvate transporter; Q,
ubiquinone; Sugar-P, sugar phosphates.
(Lemaitre and Hodges, 2006). At least three NAD-IDH
genes have been found in Nicotiana tabacum (tobacco), with
a single gene encoding a ‘catalytic’ subunit and two genes
encoding ‘regulatory’ subunits (Lancien et al., 1998). To
obtain a catalytic activity, the plant NAD-IDH must contain
at least one of each subunit type. This has been shown by the
complementation of yeast IDH mutants (Lancien et al., 1998;
Lemaitre et al., 2007), the production of recombinant
tobacco IDH proteins in bacterial cells (Lemaitre, 2005), and
the analysis of Arabidopsis knock-out mutants (Lemaitre
et al., 2007). On the other hand, NADP-IDH is homomeric
(composed of a 47 kDa subunit). In Arabidopsis, only three
genes exist for four isoenzymes with the cytosolic and
peroxisomal forms encoded by their own gene (At1g65930
and At1g54340, respectively) while a third gene (At5g14590)
encodes both the mitochondrial and plastidial isoforms
(McKinnon et al., 2009; Fig. 2). To date, the factors that
regulate the proportion of each isoform within a given
organelle are unknown.
In the literature two hypotheses have been put forward
concerning the involvement of different IDH enzymes in GS/
GOGAT functioning. It was proposed that mitochondria are
the source of 2-OG, through NAD-IDH (Miflin and Lea,
1980). In this scenario, 2-OG would leave the mitochondria,
probably via a dicarboxylate-tricarboxylate carrier named
DTC (Picault et al., 2002), to be imported into the plastids,
where the leaf GS/GOGAT cycle is located, by a transporter
named DiT1 (Weber et al., 1995; Fig. 1). The second
hypothesis involves the cytosolic NADP-IDH (Chen and
Gadal, 1990). In this case, citrate is exported from the
mitochondria (again by DTC) and transformed to isocitrate
via an aconitase and then into 2-OG by the cytosolic NADPIDH (Fig. 1). According to this second scenario, 2-OG of
cytosolic origin would again be imported into the plastids via
DiT1 in exchange for malate. A second transporter, DiT2,
exports glutamate made in the plastid, again in exchange for
malate (Renné et al., 2003). While it used to be thought that
the provision of carbon skeletons for the GS/GOGAT cycle
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Fig. 2. Genes encoding isocitrate dehydrogenase (IDH) in Arabidopsis and subcellular localization of the encoded proteins. For the six
NAD-IDH genes, ‘pg’ indicates pseudogene, ‘r’ regulatory subunit, and ‘c’ catalytic subunit. At least one catalytic and one regulatory
subunit are required to form a functional NAD-IDH.
could be dominated by one of these two routes, the data
below will show that investigations over many years have
failed to produce such evidence and that the principles
governing the flux between the two pathways is still unclear.
NAD-IDH and ammonium assimilation
Are mitochondria the source of the 2-OG required for GS/
GOGAT functioning? This organelle contains both NADIDH and NADP-IDH, but Miflin and Lea (1980) proposed
that NAD-IDH is responsible for making 2-OG required
for ammonium assimilation. To date, this role for NADIDH remains under debate since the TCA cycle enzyme is
still poorly characterized.
The best characterized NAD-IDH is the yeast enzyme.
Two genes encode the IDH1 and IDH2 subunits required for
an active enzyme composed of four IDH1–IDH2 dimers
(Panisko and McAlister-Henn, 2001). Its essential role in the
TCA cycle was shown by the ‘acetate’ phenotype of yeast
NAD-IDH mutants. However, such mutants were not
glutamate auxotrophs due to the presence of the cytosolic
and mitochondrial NADP-IDH enzymes (Zhao and McAlister-Henn, 1996). Mammalian NAD-IDH is also heteromeric
and three subunits exist (a, b, and c) with the a subunit being
equivalent to the yeast IDH2 ‘catalytic’ subunit. Recombinant mammalian NAD-IDH composed of all three subunits
showed the highest activity, although a combination of
NAD-IDHa with one of the other two subunits was active
(albeit 10-fold lower) (Kim et al., 1999). In plants, NADIDH shows a low measurable in vitro activity and the
complex is unstable, thus hampering our understanding of
its function in plant systems. As in the yeast and mammalian
systems, plant NAD-IDH activity requires at least a ‘catalytic’ and a ‘regulatory’ subunit for enzymatic activity
(Lancien et al., 1998).
Evidence for the role of NAD-IDH in ammonium assimilation is scarce. NAD-IDH transcript levels were found to
increase when N-starved tobacco plants were resupplied with
nitrate or ammonium. These changes were co-ordinated with
the expression of other TCA cycle genes (citrate synthase and
aconitase but not fumarase) and N-assimilatory genes (NR
and GS). Since the response kinetics were correlated to the
nitrate assimilatory capacity of each organ, it was proposed
that the nitrate status or metabolites associated with its
metabolism could be acting as signals (Lancien et al., 1999).
NAD-IDH expression and localization have been investigated
in rice roots with respect to N-nutrition and compared with
those of NADH-GOGAT and GDH. Only IDHa and IDHc.1
transcripts (encoding a catalytic and a regulatory subunit)
responded to the different N-regimes while IDHc.2 and GDH
did not. Under ammonium nutrition, NAD-IDH protein colocalized with NADH-GOGAT in the epidermis and the
exodermis, suggesting that it could play a role in Nassimilation (Abiko et al., 2005). Recently, tomato antisense
lines with a reduced NAD-IDH activity of around 40% were
shown to have a modified capacity to assimilate nitrate, as
judged by an increase in leaf nitrate levels and reduced amino
acid levels. The NAD-IDH mutants contained lower NADH
(and NADPH levels) in the leaves, which could impact on NR
activity. These mutants also exhibited a reduced TCA cycle
flux, decreased starch levels, less chlorophyll, and lower
organic acid amounts that suggest an N-limitation. However,
2-OG levels were not affected and plant growth was not
significantly altered. Interestingly, the mutation did affect
Respiration and nitrogen metabolism | 1471
tomato fruit yield. Although fruits were smaller and had
a lower fresh weight, fruit number was not modified
(Sienkiewicz-Porzucek et al., 2010).
A number of observations, however, argue against a
mitochondrial NAD-IDH origin for the 2-OG used to fuel
the GS/GOGAT cycle. NAD-IDH exhibits a low measurable activity in vitro and relatively high Km values for
isocitrate (280–850 lM) and NAD (150–800 lM) (see
Gálvez et al., 1999, for more details) when compared to
NADP-IDH (see below). Furthermore, the enzyme is
inhibited by NADH (Ki¼70–420 lM) which could accumulate in leaf mitochondria in the light via the action of the
photorespiratory enzyme, GDC. However, this scenario
may not take place because GDC is also inhibited by
NADH (Ki¼15 lM; Oliver, 1994). Of course, the in planta
NAD-IDH activity is perhaps higher than that measured
in vitro and NADH might not accumulate due to the need
to channel reducing power to the cytosol and the peroxisomes via a malate/oxaloacetate shuttle driven by the
activity of a dicarboxylate carrier as described by Palmieri
et al. (2008). In illuminated leaves, TCA cycle decarboxylations can be reduced by 80% and the decarboxylation
reaction of the pyruvate dehydrogenase is decreased by 30%
when compared to dark respiration (Tcherkez et al., 2008). It
is possible that this activity is not sufficient to make 2-OG
for N-assimilation. However, in photosynthetic tissues, the
activity of a complete TCA cycle appears to be reduced and
a non-cyclic TCA pathway is probably more important in the
light (illuminated leaves) due to the transport of organic acids
out of the mitochondria (Tcherkez et al., 2009; Sweetlove
et al., 2010). Recently, experiments using isotopic doublelabelling (13C/15N) and NMR analyses have indicated that the
carbon skeletons used for day N-assimilation originate from
stored organic acids (probably malate or citrate) made during
the night period (Gauthier et al., 2010).
The study of three Arabidopsis knock-out mutants for
different NAD-IDH subunits (encoded by At5g03290,
At4g35260, and At2g17130) also suggests that NAD-IDH is
not essential in producing 2-OG for N-assimilation. Under
standard, non-limiting growth conditions, the mutants,
which had only 8%, 40% or 57% of normal NAD-IDH activity in their rosette leaves, did not show any growth or developmental phenotype. However, the mutants did contain
reduced amounts of certain amino acids in their leaves but
this did not include Gln and Glu. Interestingly, the content
of NAD+ and NADPH was reduced by the modified NADIDH activity thus indicating a perturbation in cell redox level
(Lemaitre et al., 2007).
NADP-IDH and ammonium assimilation
Although an NADP-IDH is found in plastids (Gálvez et al.,
1994), the principal site of ammonia assimilation, it was
proposed by Chen and Gadal (1990) that the cytosolic
isoform is important in producing the 2-OG for amino acid
biosynthesis. As yet, no convincing evidence supporting this
hypothesis has been reported in the literature although a
number of observations have been interpreted in its favour.
Cytosolic NADP-IDH is the major isoform in green leaves.
For example, it accounts for 95% of the total tobacco leaf
NADP-IDH activity (Gálvez et al., 1994). It has been shown
to be the predominant NADP-IDH in 15 plant species (Chen,
1998) including tomato fruit (Gallardo et al., 1995), potato
(Fieuw et al., 1995) and it is the only detectable activity in pine
cotyledons (Palomo et al., 1998). These observations consolidate the idea that the cytosol has the capacity to be the
predominant source of 2-OG in the leaf. All NADP-IDH
isoforms have similar low Km values for isocitrate (11–80 lM)
and NADP (4–16 lM) that reflect their highly conserved
protein sequences (see Gálvez et al., 1999, for more details).
A number of data have linked cytosolic NADP-IDH to
amino acid biosynthesis. An analysis of NR-deficient tobacco
plants accumulating both nitrate and 2-OG, showed an
increase in cytosolic NADP-IDH transcript levels and a number of N-assimilation and organic acid-metabolism genes,
while the starch-biosynthesis pathway enzyme, ADP-glucose
pyrophosphorylase, was down-regulated. These observations
were interpreted as a functional co-ordination of a cytosolic
NADP-IDH 2-OG pathway with that of N-assimilation
(Scheible et al., 1997). This was in agreement with the preferential export of citrate from malate-fed intact mitochondria
extracted from photosynthetically active tissues (Hanning
et al., 1999). In pine, a correlation was found between
cytosolic NADP-IDH and GS transcripts during seed germination and with GS and Fd-GOGAT transcripts during
chloroplast biogenesis. However, such a correlation was not
conserved during the advanced stages of cotyledon development, thus leading the authors to suggest that, in pine,
cytosolic NADP-IDH had several functions (Palomo et al.,
1998). A similar conclusion was reached when cytosolic
NADP-IDH expression was examined in potato (Fieuw et al.,
1995). The modification of NADP-IDH transcript and activity
levels by light and mimicked by nitrate and sucrose (as also
observed for N metabolism genes: Stitt, 1999) suggested a role
in N-assimilation. Induced leaf senescence also led to an increase in NADP-IDH activity and stable NADP-IDH protein
levels while many other protein levels decreased. Stable leaf
NADP-IDH transcript levels were also reported in ageing
tobacco plants (Masclaux et al., 2000). These observations
were interpreted in terms of a role for cytosolic NADP-IDH
in the cycling, redistribution, and export of amino acids during
leaf senescence. A similar function was attributed to this
NADP-IDH isoform during tomato fruit ripening due to a 2–
3-fold increase in NADP-IDH activity associated with glutamate accumulation (Gallardo et al., 1995).
Despite these elements, as with NAD-IDH, studies using
transgenic plants (either antisense or T-DNA knock-out
mutants) with reduced or absent cytosolic NADP-IDH activity were not able to show an essential role for this enzymatic activity in N-assimilation. Potato (Kruse et al.,
1998) and tobacco (S Gálvez and M Hodges, unpublished
data) retaining less than 10% of their total NADP-IDH activity, exhibited no deleterious growth phenotype. A detailed analysis of the antisensed potato plants showed no
noticeable alterations in either C or N metabolism while
amino acid content was unchanged. Recently, Arabidopsis
1472 | Foyer et al.
mutants lacking cytosolic NADP-IDH activity have been
isolated and characterized (Mhamdi et al., 2010). Three
allelic T-DNA knock-out lines grew normally and did not
show any significant developmental phenotype, although
plants did appear to be slightly smaller than their wild-type
counterparts. Metabolic profiling by GC-TOF-MS and
HPLC revealed that the loss of cytosolic NADP-IDH activity did not have a large impact on leaf compounds associated
to C and N metabolism, although fructose was reduced by
50% and citrate was increased by 50% in air-grown plants.
When grown under non-photorespiratory conditions (high
CO2), the mutant plants had decreased levels of sucrose and
malate as well as three amino acids—mainly serine but also
alanine and threonine to a lesser extent. Total amino acid
pools did not appear to be affected by the absence of
cytosolic NADP-IDH (Mhamdi et al., 2010). A recent study
of tomato plants with only partial decreases in cytosolic
NADP-IDH activity reported considerable shifts in metabolism characterized by decreases in TCA cycle intermediates,
total soluble amino acids, starch, and NAD(P)H but only a
small change in plant growth (Sulpice et al., 2010).
When tobacco plants were subjected to N-starvation
followed by nitrate or ammonium resupply, cytosolic NADPIDH transcript levels were not affected (Lancien et al., 1999).
Such observations appear to be in contradiction to those made
using the tobacco NR mutants (Scheible et al., 1997) but the
differences are probably due to the different experimental
conditions and plant history, with nitrate assimilation almost
absent in the latter case but fully active in the former.
It has also been shown that cytosolic NADP-IDH is
localized mainly in the vascular tissues of higher plant
leaves (Gálvez et al., 1996) and roots (Boiffin et al., 1998).
This is not consistent with the major ammonium assimilatory site. Interestingly, vascular tissues do contain cytosolic
GS (GS1) and NADH-GOGAT and, therefore, the presence of cytosolic NADP-IDH suggests a role in plant
metabolic functions associated with N-metabolism. In pine,
the spatial expression of NADP-IDH was not found to
coincide with that of other key N-assimilatory enzymes, and
it was concluded that NADP-IDH was a housekeeping
enzyme (Pascual et al., 2008). In conclusion, it is probable
that no specific IDH is involved in the production of 2-OG
for N-assimilation. A similar situation has been found in
yeast cells: indeed, glutamate auxotrophy requires the
absence of NAD-IDH and the mitochondrial and cytosolic
NADP-IDH isoenzymes (Zhao and McAlister-Henn, 1996).
NADP-IDH in redox homeostasis and related processes
Over the last decade, there are ever-increasing reports,
especially in mammalian cells, that NADP-IDH could have
a major role in the production of NADPH required for
protection against oxidative stress. Mitochondrial NADPIDH has been shown to be important in the cellular response
to heat shock (Shin et al., 2007), cadmium (Kil et al., 2006),
and other treatments that lead to an oxidative stress and
eventually to apoptosis (cell death) in different types of
mammalian cell (Jo et al., 2001). In the case of a Cd stress,
even though Cd binds to mitochondrial NADP-IDH and
induces a loss of activity, it was shown that mitochondrial
NADP-IDH activity was important in protecting glutaredoxin
from Cd and thereby reducing cell damage (Kil et al., 2006).
Resistance against the adverse effects of selenium has also
been shown to be decreased in cells with reduced mitochondrial NADP-IDH levels (Kil et al., 2010). IDP1 (mitochondrial NADP-IDH) was found to be important in producing
NADPH in the fungal pathogen Cryptococcus neoformans,
thus protecting it against nitrosative stress and mitochondrial
damage (Brown et al., 2010). Cytosolic NADP-IDH has also
been shown to play an important role in the protection against
oxidative stress in mice renal cells (SH Lee et al., 2010). The
analysis of yeast idp2 (cytosolic NADP-IDH) mutants showed
that this isoform also has an antioxidant role. The effects were
more severe in idp2/gluc-6-PDH double mutants, thus suggesting that cytosolic NADP-IDH is not the only enzyme that
produces NADPH for such antioxidant functions (Minard
and McAlister-Henn, 2001). In plants, it has been found that
mitochondrial NADP-IDH is involved in the reductive
activation of alternative oxidases. Indeed, the overexpression
of this NADP-IDH isoform led to the presence of more
reduced, active alternative oxidase subunits (Gray et al., 2004).
A role for cytosolic NADP-IDH in plants was suggested from
the recent study of Arabidopsis knock-out lines. When crossed
with a catalase (cat2) mutant that induces an increased
oxidative stress triggered by enhanced H2O2 availability,
knockout mutations for cytosolic NADP-IDH caused more
marked accumulation of oxidized glutathione and enhanced
pathogen-related responses compared with the cat2 parent line
(Mhamdi et al., 2010). Pathogenesis-related responses were
also observed in single NADP-IDH mutants. These results
suggest that cytosolic NADP-IDH could play a role in redox
or other signalling linked to pathogen responses (Mhamdi
et al., 2010).
In mammalian systems, it appears that both mitochondrial
and cytosolic NADP-IDHs are protected against oxidative
stress by a process called protein S-glutathionylation. Indeed,
NADP-IDH activity is susceptible to inactivation by a number
of thiol-modifying reagents. It was shown that mitochondrial
NADP-IDH can become glutathionylated on Cys269, thus
leading to an inactive enzyme. However, it was reactivated by
glutaredoxin2 in the presence of GSH (Kil and Park, 2005).
Glutathionylated mitochondrial NADP-IDH was found to be
less susceptible to ROS-induced proteolysis during oxidative
stress. Similar observations have been made concerning the
cytosolic NADP-IDH in mice kidney cells where Cys269 was
also shown to become glutathionylated (Shin et al., 2009). In
plant systems, there is no reported evidence of such a process
occurring, although plant NADP-IDH proteins do contain the
equivalent Cys residue.
Regulation of the oxidative pentose
phosphate pathway
The metabolic pathway of N assimilation involves the
reduction of nitrate to nitrite by the cytosolic enzyme
Respiration and nitrogen metabolism | 1473
nitrate reductase (NR) at the expense of NADH produced
from malate shuttled from the mitochondrion or chloroplast
(Foyer and Noctor, 2002). In leaves, nitrite then enters the
chloroplast either as the neutral acid (HNO2) or via a
transporter (Sugiura et al., 2007), where it is reduced by the
ferredoxin system to ammonium with the evolution of 1.5 O2
per nitrite reduced. Thereafter, the ammonium is combined
with 2-OG in the GS/GOGAT system to produce glutamate.
While many higher plant species predominantly undertake
this pathway in leaves others assimilate nitrate mainly in the
root and many species such as white lupin and soybean use
both organs with about 45% of the nitrate assimilation
taking place in the roots (Cen et al., 2001; Cen and Layzell,
2003). Estimates of carbon partitioning in roots suggest that
about 15% of root carbon catabolism is coupled to ammonium assimilation under ammonium nutrition, whereas
under nitrate nutrition about 5% of the root carbon
catabolism is coupled to nitrate absorption from the soil,
15% to nitrate assimilation, and 3% to ammonium assimilation (Bloom et al., 1989, 1992, 2002).
The reducing power provided by photosynthesis drives
primary nitrogen assimilation in green tissues in the light, but
in the dark and in non-green tissues, the necessary reducing
power is supplied by the oxidative pentose phosphate pathway (OPPP; Bowsher et al., 1992; Esposito et al., 2003). The
interdependency of nitrogen assimilation and respiratory
carbon assimilation in terms of reducing power and carbon
metabolism has been studied intensively for many years
(Emes and Fowler, 1979; Bowsher et al., 1989, 1993;
Wright et al., 1997). While the complete OPPP is found in
plastids, the non-oxidative reactions also occur in the cytosol.
The first committed step of the pathway is catalysed by
glucose 6-phosphate dehydrogenase (G6PDH), which is
considered to be the major control point of the pathway and
6-phosphogluconate dehydrogenase (6PGDH). Together,
these enzymes convert glucose 6-phosphate (Glc6P) to
ribulose 5-phosphate with the production of two molecules
of NADPH. Of the six G6PDH gene family members in A.
thaliana, four are predicted to encode plastidial proteins
(Wakao and Benning, 2005). At the protein level, two
plastidial isoforms, P1-G6PDH and P2-G6PDH, have been
the most studied (Kruger and von Schaewen, 2003).
All eukaryotic G6PDHs studied to date are subject to
feedback inhibition by NADPH and they are therefore
presumed to act as cellular redox sensors. While G6PDH
transcripts and activity have been shown to be induced by
nitrogen in various plants, the cytosolic G6PDH forms are
largely insensitive to nitrogen status and to thioredoxin
(TRX)-mediated redox regulation (Fickenscher and Scheibe,
1986). By contrast, the plastid isoenzymes are subject to both
forms of control. P1-G6PDH is controlled by light-regulated
reductive inactivation through the stromal TRX system and
by changes in the stromal NADPH/NADP ratio (Wenderoth
et al., 1997) but it is not greatly affected by nitrogen. The P2G6PDH form is directly responsive to tissue nitrogen status
and is more resistant to changing NADPH levels than P1G6PDH (Esposito et al., 2001, 2005). Unlike leaf FdGOGAT activities, which are enhanced in the presence of
nitrogen, root Fd-GOGAT activity and protein are substantially unchanged by nitrogen availability. The induction
of the P2-G6PDH form by nitrogen leading to enhanced
P2-G6PDH-mediated OPPP activity is considered to be
particularly important in driving root nitrogen assimilation
linked to NADH-GOGAT (Esposito et al., 2001, 2005).
While the oxidation of Glc6P by the OPPP in the roots is
capable of supporting nitrite reductase (NiR; Bowsher et al.,
1989) and GOGAT (Bowsher et al., 1992) activities alone,
the capacity of the pathway is insufficient to support the
simultaneous operation of both NiR and GOGAT (Bowsher
et al., 2007). The capacity of the OPPP in root plastids is
therefore insufficient to meet the demands of nitrogen assimilation and the flux through the OPPP constrains the
nitrogen assimilation ability of root plastids (Bowsher et al.,
2007). The oxidation of Glc6P, rather than the activities of
nitrogen assimilation enzymes, limits root nitrogen assimilation. If the requirements of primary nitrogen assimilation are
combined with the reductant requirements of other pathways
in non-photosynthetic plastids, then it is likely that the rate
of carbohydrate oxidation by the OPPP activity represents a
major limitation. G6PDH is generally considered to be an
important source of NADPH in non-photosynthetic tissues
other than roots, particularly those that produce fatty acids
at high rates, such as oil seeds. However, while the loss of
cytosolic G6PDH adversely affected the metabolism of developing seeds it appeared to do so by increasing the
availability of carbon substrates for the synthesis of storage
compounds rather than by decreasing the NADPH supply
for fatty acid synthesis (Wakao et al., 2008).
Plastidial G6PDH belongs to a group of enzymes that
respond rapidly to reductive regulation by the chloroplast
TRX system. This group of chloroplast enzymes also includes
the ATP-synthase and aggregates of phosphoribulokinase and
non-regulatory glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) that are linked by the redox-regulated CP12
protein. G6PDH is inactivated by TRX in the light, thereby
turning off the OPPP and preparing acceptor molecules for
carboxylation. The redox potentials of the different enzymes
would suggest that this process would occur before the
reductive activation of ribulose-1,5-bisphosphate carboxylase
oxygenase (RuBisCo) activase and the regulatory form of
GAPDH that would initiate the carboxylation reactions that
begin the reductive pentose phosphate pathway (Calvin
Cycle). The reductive inactivation of the G6PDH forms is
presumed to prevent unnecessary carbon oxidation when
there is sufficient NADPH generation by photosynthesis. It is
perhaps not surprising that G6PDH transcript and activity
levels increase upon exposure to oxidative stresses such as
that induced by methyl viologen, and by inhibitors of the
photosynthetic electron transport system.
In addition to the regulation of the activities of G6PDH
and thiol-modulated reductive pentose phosphate pathway
enzymes, the plastid TRX system also participates in the
control of starch turnover through reductive activation of
ADP-glucose pyrophosphorylase (AGPase). The AGPase
from potato tubers and leaf chloroplasts is activated by
TRXs f and m allowing the enzymes of the reductive pentose
1474 | Foyer et al.
phosphate pathway and of starch synthesis to be regulated in
a co-ordinate manner (Hendriks et al., 2003; Kolbe et al.,
2005). A second TRX system, involving NADP-TRX reductase C (NTRC), whose activity is triggered in response to
sucrose, also operates in plastids. The NTRC system facilitates
redox regulation of AGPase in roots and in leaves and other
green tissues in the dark (Michalska et al., 2009). The
combined operation of the NTRC and Fd/TRX systems may
provide a mechanistic link between reactions in the chloroplasts and the amyloplasts and other plastids of heterotrophic
tissues through the sucrose triggered activation of AGPase.
Redox activation of AGPase is induced by sucrose and other
sugars in leaves in both the light and dark (Hendriks et al.,
2003). In this way, starch synthesis in organs far removed
from the leaves is responsive to signals from the chloroplasts
that contain information regarding the carbon status of the
plant (Kolbe et al., 2005). NTRC is considered to protect leaf
enzymes against enhanced cellular oxidation, particularly
under low light conditions or when leaves are subjected to
prolonged darkness, but there is as yet no information on how
this system might interact with the TRX-dependent oxidative
activation of G6PDH and the functioning of the OPPP in
chloroplasts in the dark or in root plastids to allow the
provision of sufficient carbon for respiration.
Regulation of the respiratory electron
transport chain
The plant respiratory electron transport chain consists of four
multi-subunit oxidoreductases, Complexes I to IV, and the
ATP synthase complex (Complex V) with additional oxidoreductases. The alternative oxidase (AOX) allows electrons to
bypass Complexes III to IV of the cytochrome pathway and
provides a mechanism for oxidation of the ubiquinone pool.
This is considered to prevent the build-up of electrons within
the electron transport system and to minimize the risk of
electron donation to oxygen and superoxide formation. In
addition, there are up to four different ‘rotenone-insensitive’
type II NAD(P)H dehydrogenases, which circumvent Complex I of the electron transport chain. All the complexes
apart from succinate dehydrogenase (Complex II) participate in the formation of supercomplexes, which have the
potential to add another layer of complexity to the regulation of respiratory electron transport. For example,
supercomplexes involving complexes I, III and IV might
operate in a single functional unit allowing a direct electron
channel or path from NADH to cytochrome c (Eubel et al.,
2004). This type of supercomplex has been called a ‘respirasome’ because it can carry out respiratory electron flow in
the presence of ubiquinone and cytochrome c in an
autonomous manner (Dudkina et al., 2005). Supercomplex
formation might also assist in the regulation of alternative
respiratory pathways via electron channelling. Like the
‘alternative’ and accessory components, such supercomplexes
confer energetic flexibility on the system by allowing adjustments in ADP:O ratios (Rasmusson et al., 2004). Interestingly, comparisons of the root and shoot mitochondrial
proteomes have failed to reveal any major differences in the
abundance of mitochondrial electron transport chain components. In marked contrast, the abundance of enzymes
involved in the TCA cycle and in photorespiration were
different in the root and shoot mitochondrial proteomes (CP
Lee et al., 2010). Such results suggest that changes in the
abundance of proteins involved in the TCA cycle and in
photorespiration can be sufficient to allow adaptation of
mitochondria to leaf and root metabolism without changes in
the content or composition of components of the electron
transport system (CP Lee et al., 2010).
The type II NAD(P)H dehydrogenases and AOX are
considered to function in higher plants as a defence against
metabolic fluctuations. It is perhaps not surprising therefore
that the expression of the NAD(P)H dehydrogenases and
AOX genes is influenced by the availability of nitrate and
ammonium. In Chlamydomonas reinhardtii, for example, the
expression of Aox1, the major AOX, is strongly downregulated by ammonium and stimulated by nitrate in line
with changes in the rate of respiration (Baurain et al., 2003).
In A. thaliana, the expression of the NAD(P)H dehydrogenase and AOX genes was decreased in the presence of nitrate
and increased by ammonium (Escobar et al., 2006). The
switch from nitrate to ammonium enhanced respiration rates
and increased AOX activity and protein as well as calciumdependent external NADH oxidation, trends consistent with
an increased capacity of respiratory bypass pathways after
switching from nitrate to ammonium (Escobar et al., 2006).
The impact of the mitochondrial electron transport chain
on photosynthetic and respiratory metabolism has long
been studied in leaves and protoplasts using pharmacological methods (Krömer et al., 1988, 1993; Raghavendra and
Padmasree, 2003). Recent years have witnessed several
analyses of such questions using reverse genetics approaches
(Noctor et al., 2007). For example, Aox1a knockout mutants
accumulate sugars but have lower organic and amino acids
compared with the wild type, consistent with the view that, in
the absence of AOX and alternative non-phosphorylating
respiration, the respiratory chain cannot alleviate the tight
constrictions of adenylate control (Giraud et al., 2008).
Knockouts in Complex I and uncoupling proteins have been
shown to exert a strong influence on photosynthesis (Dutilleul et al., 2003; Sweetlove et al., 2006), as do decreases in
components of the TCA cycle (Carrari et al., 2003; NunesNesi et al., 2005, 2007).
Over the last 10 years, a range of studies on Complex I
mutants has unequivocally demonstrated that the path of
NADH oxidation, through either Complex I or the alternative
NAD(P)H dehydrogenases, has a significant impact on
nitrogen metabolism and carbon/nitrogen interactions. Two
mutants have proved to be particularly useful tools in revealing this relationship. These are the Nicotiana sylvestris
CMSII mutant, which carries a mutation in the mitochondrial
nad7 gene (Gutierrez et al., 1997; Pineau et al., 2005), and the
A. thaliana ndufs4 that lacks the fragment S subunit 4 18 kDa
subunit of Complex I. As a consequence of these mutations,
both lines are completely deficient in respiratory Complex I.
The Complex I-deficient mutants grow more slowly than their
Respiration and nitrogen metabolism | 1475
respective wild types but they remain viable because respiration can be sustained through the alternative NAD(P)H
dehydrogenases, albeit with constitutively lower phosphorylation efficiencies (Sabar et al., 2000; Meyer et al., 2009). The
loss of Complex I has a marked influence on cellular redox
status, particularly the NAD plus NADH pool, which is
increased as a result of Complex I deficiency (Dutilleul et al.,
2005; Hager et al., 2010). The enhanced availability of
NADH in the Complex I mutants has important implications
for the regulation of nitrogen assimilation and metabolism
(Dutilleul et al., 2005; Meyer et al., 2009). The switch from
NADH oxidation by Complex I to the alternative NAD(P)H
dehydrogenases results in a nitrogen-rich phenotype with
general increases in amino acids and organic acids and
a decrease in starch (Dutilleul et al., 2005; Meyer et al.,
2009; Hager et al., 2010).
Higher nitrate assimilation rates were observed in the
absence of a functional Complex I, together with altered C/N
status (Dutilleul et al., 2005; Pellny et al., 2008). Metabolomics analyses of the N. sylvestris and A. thaliana Complex
I mutants have consistently shown that nitrogen-rich metabolites accumulate in the leaves (Dutilleul et al., 2005; Meyer
et al., 2009; Hager et al., 2010). The effect of Complex
I deficiency is most pronounced in leaves in the dark, not
least because the mutants cannot shut down nitrate assimilation at night (Dutilleul et al., 2005). Moreover, mitochondrial
respiration plays a major role in controlling metabolic fluxes
in the dark. Other features consistent with a high tissue
nitrogen status are found in the Complex I mutants, such as
a repression of lateral root production and altered biomass
partitioning between roots and shoots in response to nitrogen
availability (Pellny et al., 2008).
Mitochondrial redox cycling as a key player
determining the rate of nitrate assimilation
In addition to integration with respiratory C flow, N
assimilation in the leaves of C3 species is also intimately
associated with photorespiratory C flow, an interaction that
further complicates the photosynthetic C and N interaction
(Stitt et al., 2002; Foyer et al., 2009). Under most conditions,
the photorespiratory cycle is much more important and rapid
than primary nitrate reduction. Photorespiration can influence
N assimilation in several ways (Foyer et al., 2009). Ammonia
liberated during the conversion of Gly to Ser in leaves is recycled through Gln and Glu, and this process probably occurs
through the same GS and GOGAT isoforms as those involved
in primary ammonia assimilation (Hirel and Lea, 2002).
Photorespiration also involves reductant cycling. NADH is
produced by Gly oxidation in the mitochondria. While
stoichiometrically equal amounts of NADH are required
during steady-state operation of the photorespiratory pathways for glycerate synthesis in the peroxisomes (Hanning and
Heldt, 1993), the enhanced generation of light-dependent
reductant in both the chloroplasts and mitochondria could
contribute to increasing cytosolic NADH concentrations.
These concentrations are rather low during most conditions,
and the cytosolic NAD pool, estimated at about 0.7 mM, has
been considered to be more than 99% NAD+ (Heineke et al.,
1991). For comparison, the Km NADH of NR is 1–5 lM
(Campbell, 1999).
For the above reasons, the ability of chloroplasts or
mitochondria to deliver reducing equivalents to the cytosol
could influence the rate of N assimilation (Fig. 3; Krömer
and Heldt, 1991; Hanning and Heldt, 1993). Low photosynthesis rates could limit NR activity through decreased
reductant availability, as well as through post-translational
inactivation (Kaiser et al., 2000, 2002). Nitrate reduction is
significantly stimulated by anoxia, conditions that are
expected to favour export of mitochondrial reductant to the
cytosol. Such export is likely to be favoured in conditions in
which the complex redox cycling of photorespiration is active
and GDC activity is significant (Fig. 3). Indeed, photorespiration has been reported to be necessary for optimal rates of
nitrate assimilation in several crop species (Rachmilevitch
et al., 2004; Bloom et al., 2010). This may involve improved
availability of reductant in both the cytosol and chloroplast
when photorespiration is favoured (Bloom et al., 2010).
Fundamental mechanistic questions remain, however, concerning the links between photorespiration, respiration, and
nitrogen assimilation. Delivery of reductant from the chloroplast to the cytosol could also be favoured by the operation
of triose-phosphate/3-phosphoglycerate shuttles (Fig. 3). This
is predicted to be favoured by high photosynthesis rather
than high photorespiration, and an important role for such
shuttles is consistent with some literature observations
(Kaiser et al., 2000). Assuming that triose-phosphate is reoxidized through the classical glycolytic NAD-dependent
glyceraldehyde-3-phosphate dehydrogenase, the operation of
this shuttle would augment the production of cytosolic
NADH above that linked to triose phosphate metabolism
through to phosphoenolpyruvate (Fig. 1). By contrast,
a requirement of high rates of photorespiration for optimal
nitrogen assimilation rates (Rachmilevitch et al., 2004;
Bloom et al., 2010) could be explained by enhanced redox
transfer to the cytosol through the chloroplast envelope or
mitochondrial malate/oxaloacetate shuttles (Fig. 3).
An important point concerning amino acid synthesis is
that this process requires simultaneous C oxidation and
nitrate reduction. Thus, any perturbation of redox cycling
could radically alter C/N partitioning through concerted and
opposing effects on the two processes. Organic acid synthesis
may also be subject to redox modulation in the C/N
interaction because, as discussed in the first part of this
review, the production of compounds such as 2-OG requires
oxidation through respiratory pathways involving the cytosol
and mitochondria (Foyer et al., 2003, 2009; Hodges, 2002;
Noctor et al., 2007).
It remains unclear whether part of the Complex I deficiency
on nitrogen metabolism is mediated through effects on
photorespiration. The mitochondrial electron transport chain
oxidizes NADH generated by various soluble mitochondrial
enzymes, notably GDC and TCA cycle dehydrogenases
(Rasmusson et al., 2004; Noctor et al., 2007). Mitochondria
lacking Complex I have a decreased capacity for glycine
1476 | Foyer et al.
Fig. 3. Major redox shuttles potentially linking mitochondria and chloroplasts to the generation of NADH in the cytosol, where nitrate
reductase (NR) is located. Numbered reactions are catalysed by (1) PGA kinase; (2) NADP- or NAD-linked glyceraldehyde-3-phosphate
dehydrogenase; (3) NADP- or NAD-linked malate dehydrogenase; (4) glycine decarboxylase. Abbreviations: FNR, ferredoxin-NADP
reductase; bPGA, 1,3-bisphosphoglycerate; 3-PGA, 3-phosphoglycerate. Other abbreviations as for Fig. 1.
oxidation (Sabar et al., 2000), possibly due to increased
redox control over glycine decarboxylase. Despite this, the
glycine/serine ratios were not increased in leaves of the
Complex I mutants in air (Dutilleul et al., 2003, 2005),
although the decreased mesophyll conductance might indicate higher rates of photorespiration (Priault et al., 2006).
Mitochondrial targets for the enhancement
of nitrogen use efficiency (NUE)
Recent increases in our understanding of the interactions
between respiration and nitrogen assimilation have revealed
that mitochondrial metabolism is a potential target for
enhancing NUE. For example, recent evidence suggests that
the effects of Complex I mutations in different species show
a strong similarity to each other in terms of effects on nitrogen
metabolism and the accumulation of amino acids. These
results demonstrate that low Complex I activities favour Nrich phenotypes. Moreover, limitations at the level of the
OPPP capacity rather than nitrogen assimilation itself have
been identified in roots. Therefore, manipulation of the
different G6PDH isoforms to enhance flux through the OPPP
in roots is a potential new target for enhancing NUE.
Of the new potential targets for improved NUE, increasing
NAD plus NADH availability by manipulating the relative
capacities of Complex I and the type II NAD(P)H dehydrogenases is attractive because the availability of NADH exerts
a strong influence on nitrogen assimilation and amino acid
accumulation. The relationship between Complex I and the
type II NAD(P)H dehydrogenases is a flexible parameter and
potentially open to manipulation. The factors that relate
perturbation of the respiratory electron transport chain to
high amino acid contents remain to be clearly identified. This
could partly occur through stimulation of nitrate assimilation
but other factors may also be involved. There is a close but
not unbreakable correlation between amino acids synthesized
through different pathways (Noctor et al., 2002; Fritz et al.,
2006). Post-translational control of protein redox state could
be one influential mechanism. In this connection, it is
intriguing that plants engineered to have high glutathione
contents through chloroplastic overexpression of the glutathione synthesis pathway showed enhanced accumulation of
several amino acids (Noctor et al., 1998).
Recent studies have highlighted the central role for
pseudo-response regulators in controlling metabolic homeostasis in the TCA cycle (Fukushima et al., 2009; Nakamichi
et al., 2005). Moreover, the teosinte branched1, cycloidea,
PCF (TCP) family of transcription factors and the site II
cis-acting regulatory elements to which they bind have been
shown to link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis
thaliana (Giraud et al., 2010). These transcription factors
appear to be central regulators in circadian clock input
sites, the clock oscillator itself, and of clock output sites,
directing and regulating the expression of genes encoding
cellular energy metabolism components, particularly in
mitochondria (Giraud et al., 2010). The demonstration of
the involvement of these transcription factors in growth and
developmental processes not only provides a molecular link
co-ordinating metabolism, growth, and development but it
offers new potential targets with regard to enhancing
metabolism. For example, the site II elements in the
promoter regions of genes encoding mitochondrial, plastid,
and peroxisomal proteins provide a direct mechanism for
the co-ordination of the circadian clock with the expression
Respiration and nitrogen metabolism | 1477
of genes involved in a variety of organellar functions, including energy metabolism.
Analyses of the different IDHs in supplying 2-OG to
ammonia assimilation continue to support ideas of redundancy between the different forms (Lancien et al., 2000).
However, the recent identification of activation of pathogenesis responses in mutants lacking the cytosolic NADPdependent isoform point to new possible additional functions
(Mhamdi et al., 2010). Although the mechanisms linking loss
of cytosolic NADP-IDH to pathogen responses remain
unclear, this finding is one of several over recent years that
underline the importance of primary metabolism in pathogen
responses (Bolton, 2009). The effect may be partly linked to
redox effects mediated by NADPH or secondary effects on
respiration downstream of 2-OG. However, it is worth
noting that, as well as its role in ammonia assimilation, 2OG is a cofactor for dioxygenase enzymes that are important
in several phytohormone synthesis pathways. It remains
unclear whether 2-OG could limit such enzymes at concentrations that are sufficient for ammonia assimilation, although compartmentation is an obvious potentially
influential factor. Finally, it is important to note that 2-OG
is also an important signal metabolite that is sensed by a
protein named PII. When 2-OG is bound to this protein, PIItarget protein interactions and hence PII-regulated activities
are modified (Uhrig et al., 2009). To date, in plants, PII is
known to modify Arginine (Arg) (an N-rich amino acid)
biosynthesis by activating the N-acetyl-glutamate kinase
(Ferrario-Méry et al., 2006) and by reducing its retroinhibition by Arg (Chen et al., 2006). More recently, a role
for PII in regulating fatty acid synthesis has been indicated
by its interactions with the BCCP subunit of a plastidial
acetyl-CoA carboxylase, which decreases the activity of this
enzyme (Feria Bourrellier et al., 2010). PII mutants show
altered Arg levels in rosette leaves as well as reduced starch
and increased amino acid contents when plants are grown
under certain N-regimes, indicating a role for this protein in
C/N interactions (Ferrario-Méry et al., 2005). PII and 2-OG
levels could have a significant role in controlling the flow of
C to either N or lipid metabolic pathways depending upon
environmental conditions.
Conclusions and perspectives
Nitrogen is a major factor limiting plant growth in the field.
Crop productivity is largely dependent on high nitrogen
fertilization rates, but this practice is costly and has negative
environmental impacts. An enhanced understanding of the
mechanisms that determine NUE is required so that the
current levels of nitrogen fertilization can be decreased,
together with the identification of new targets for plant
improvement. Factors associated with carbon and nitrogen
metabolism have a close relationship to crop yields and they
are thus important current targets for improving NUE,
particularly using quantitative trait loci. To date, studies
concerning the improvement of NUE have focused largely
on the components of primary nitrogen assimilation and
associated carbon metabolism and few have looked at the
wider perspective of manipulation of energy utilization as a
further means to achieve this goal. It is suggested that
exploitation of the flexibility of respiratory pathways has
the potential to enhance NUE.
It has long been recognized that respiration and nitrogen
assimilation are intimately linked in plant cells, particularly
because of energy and metabolite requirements. However, it is
only recently that a more comprehensive understanding of the
interactions and limitations on these pathways has been
demonstrated largely through the application of -omics
technologies and forward and reverse genetic techniques.
These approaches have not only yielded valuable new insights
into the crucial role of cellular energy balance as a broker of
co-ordinate regulation but they have also provided evidence
that components of the mitochondria are exciting potential
novel targets for the enhancement of NUE. Some of the
possibilities for manipulation of the respiratory processes
have been highlighted here in order to provide a more
efficient driving force for nitrogen in plants. There is
significant potential for exerting a positive influence on NUE
by either genetic manipulation of components of respiratory
electron transport chain and/or OPPP, as well as the
identification of new molecular markers and aids to quantitative trait loci (QTL) analysis. Forward and reverse genetic
approaches, coupled to classical biochemistry and physiology,
will remain essential tools for analysis of the roles of
mitochondrial targets in improving NUE.
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