Download Use of mitochondrial electron transport mutants

Journal of Experimental Botany, Vol. 55, No. 394, pp. 49±57, January 2004
DOI: 10.1093/jxb/erh021 Advanced Access publication 17 November, 2003
FOCUS PAPER
Use of mitochondrial electron transport mutants to
evaluate the effects of redox state on photosynthesis,
stress tolerance and the integration of carbon/nitrogen
metabolism
Graham Noctor1,*, Christelle Dutilleul2, Rosine De Paepe2 and Christine H. Foyer3
1
Laboratoire Signalisation Redox, Institut de Biotechnologie des Plantes, BaÃtiment 630, Universite Paris XI,
F-91405 Orsay cedex, France
2
Laboratoire Mitochondries et MeÂtabolisme, Institut de Biotechnologie des Plantes, BaÃtiment 630, UniversiteÂ
Paris XI, F-91405 Orsay cedex, France
3
Crop Performance and Improvement, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
Received 18 June 2003; Accepted 3 October 2003
Abstract
Primary leaf metabolism requires the co-ordinated
production and use of carbon skeletons and redox
equivalents in several subcellular compartments. The
role of the mitochondria in leaf metabolism has long
been recognized, but it is only recently that molecular
tools and mutants have become available to evaluate
cause-and-effect relationships. In particular, analysis
of the CMSII mutant of Nicotiana sylvestris, which
lacks functional complex I, has provided information
on the role of mitochondrial electron transport in leaf
function. The essential feature of CMSII is the absence
of a major NADH sink, i.e. complex I. This necessitates
re-adjustment of whole-cell redox homeostasis, gene
expression, and also in¯uences metabolic pathways
that use pyridine nucleotides. In air, CMSII is not able
to use its photosynthetic capacity as well as the wild
type. The mutant shows up-regulation of the leaf antioxidant system, lower leaf contents of reactive oxygen
species, and enhanced stress resistance. Lastly, the
loss of a major mitochondrial dehydrogenase has
important repercussions for the integration of primary
carbon and nitrogen metabolism, causing distinct
changes in leaf organic acid pro®les, and also affecting downstream processes such as the biosynthesis
of the spectrum of leaf amino acids.
Key words: C/N metabolism, complex I, mitochondrial
electron transport mutants, photosynthesis, redox state,
stress tolerance.
Introduction
There is growing interest in the role of redox state in the
orchestration of plant cell metabolism and in the determination of cell fate. Even in the soluble phase of the cell,
`redox state' can mean many things, because, under most
conditions, the major soluble redox-active couples (e.g.
pyridine nucleotides, thioredoxins, glutathione, ascorbate)
are unlikely to be in equilibrium with each other (Noctor
et al., 2000). Thus, consideration of the in¯uence of redox
state depends on the redox couple under discussion. For
dehydrogenase activities, the major immediate in¯uence
will be the reduction state of NAD or NADP. For the
regulation of protein function by thiol/disuphide exchange,
the redox states of thioredoxins and/or glutathione are
likely to be most important. Other important redox-active
compounds that interact with these components are
reactive oxygen species (ROS), and their rates of production and destruction. Growing evidence demonstrates that
all the above elements are not only key to the control of
metabolism but are also sensed to initiate changes in gene
expression. The primary aim of this review is to discuss
* To whom correspondence should be addressed. Fax: +33 1 69 15 34 25. E-mail: [email protected]
Abbreviations: AOX, alternative oxidase; APX, ascorbate peroxidase; Ci, leaf intercellular CO2 concentration; CMS, cytoplasmic male sterile; GDC, glycine
decarboxylase; NDin, mitochondrial internal alternative NADH dehydrogenase; NR, nitrate reductase; 2-OG, 2-oxoglutarate; ROS, reactive oxygen species;
TCA, tricarboxylic acid; WT, wild type.
Journal of Experimental Botany, Vol. 55, No. 394, ã Society for Experimental Biology 2004; all rights reserved
50 Noctor et al.
recent evidence derived from studies of the Nicotiana
sylvestris mutant, CMSII, relating to the role of mitochondrial redox state in leaf metabolism and defence.
Leaf mitochondria play key roles in photosynthesis, e.g.
glycine decarboxylation in C3 plants and malate decarboxylation in certain C4 plants. Moreover, leaf mitochondrial
function will depend on whether the cells are photosynthetic or non-photosynthetic. Despite extensive work on
puri®ed leaf mitochondria and mitochondrial components
(Douce and Neuburger, 1989; Douce et al., 2001; Mùller
and Rasmusson, 1998; Mùller, 2001), much remains to be
elucidated concerning the role of the mitochondrial
electron transport chain in the integration of leaf metabolism (for reviews, see KroÈmer, 1995; Hoefnagel et al.,
1998; Foyer and Noctor, 2000). The key questions
addressed below are: How important is mitochondrial
electron transport in the C3 photosynthetic process? What
is the signi®cance of changes in mitochondrial redox state
for leaf redox signalling and stress tolerance? Do changes
in mitochondrial redox state impact on the co-ordination of
carbon and nitrogen assimilation in leaves? An incisive
approach to answering these questions is the analysis of
plants in which key components of the mitochondrial
electron transport chain have been manipulated genetically. Isolated in Orsay, the Nicotiana sylvestris mutant,
CMSII, is one of the few such stable mutants characterized
to date.
Phenotypic and developmental characteristics
of CMSII
Numerous studies on the consequences of genetic modi®cation of enzyme expression have emphasized the
impressive ¯exibility of leaf metabolism. It might therefore be predicted that leaves possess considerable redundancy in redox exchange between subcellular
compartments, and that the impact of non-lethal mutations
would be absorbed by metabolic adjustments, preventing
marked perturbations or phenotypic effects. The essential
feature of CMSII is a deletion mutation in the mitochondrial gene, nad7, leading to the absence of functional
complex I (Gutierres et al., 1997). Thus, the mutant has
lost the function of a major NADH sink, resulting in partial
male sterility, i.e. decreased production of pollen.
Although the mutation also causes slower shoot growth
during the vegetative phase, the ®nal biomass attained by
the mutant is not signi®cantly different from the wild type
(WT). It is therefore possible to analyse the two genotypes
at comparable stages of shoot development, and thus to
obtain information on the importance of mitochondrial
electron transport status in leaf physiology and metabolism. A comparable stage of development is achieved by
sowing CMSII seeds 3 weeks earlier than the WT. Figure 1
compares the morphology of CMSII with the WT at about
10 d prior to the stage at which sampling is performed.
Fig. 1. Photograph of Nicotiana sylvestris WT (right) and the CMSII
mutant, de®cient in mitochondrial complex I (left). Six plants of each
genotype are shown. WT and CMSII plants were germinated 9 and 12
weeks, respectively, prior to the time at which the photograph was
taken. The data discussed in this review were obtained by analysis of
plants at a developmental stage corresponding to 10 d later than that
shown in the photograph, when both genotypes are comparable in
shoot size and leaf number.
Respiratory activities of the CMSII mutant
Despite the absence of complex I function, CMSII does not
show decreased rates of leaf respiration in the dark,
suggesting that TCA cycle activity is maintained in the
mutant by complex II and alternative dehydrogenases that
bypass complex I (Sabar et al., 2000; Dutilleul et al.,
2003a). In mitochondria isolated from CMSII leaves, O2
consumption is insensitive to rotenone and ADP/O values
are signi®cantly lower than in WT mitochondria (Sabar
et al., 2000). Flux to O2 in mutant mitochondria is
probably facilitated by the increased reduction state of
mitochondrial NAD(P) pools, which allows the internal
alternative dehydrogenases to be engaged (Mùller, 2001).
At ®rst sight, therefore, the response of respiration to the
loss of complex I seems to point to a further example of
enzyme redundancy in plants. However, one of the
principal themes of this article is to show that, even if
respiration is not decreased when complex I is nonfunctional, the absence of this NADH sink has important
consequences for leaf function, and necessitates numerous
adjustments in leaf metabolism and gene expression that
uncover the potentially critical in¯uence of mitochondrial
redox status.
A key observation in isolated CMSII mitochondria is
that respiratory capacity relative to the WT is substratedependent (Sabar et al., 2000). Gly metabolism is inhibited
about 70% relative to the WT, whereas respiration of
malate, in the presence of pyruvate, is unchanged (Sabar
et al., 2000). Subsequent work showed that, in the dark,
Redox interactions in mitochondrial complex I mutants
CMSII leaf discs converted Gly to Ser slower than WT leaf
discs (Dutilleul et al., 2003a). These observations cannot
be explained by a possible absence of catalytically active
Gly decarboxylase (GDC). First, the mutant shows a
pronounced burst of CO2 evolution during the ®rst minute
following a light±dark transition and, second, Gly/Ser
ratios in the light are not increased relative to the WT
(Dutilleul et al., 2003a). Thus, the internal alternative
NADH dehydrogenase (NDin) appears to be able to
function with malate dehydrogenase and/or malic enzyme
but much less ef®ciently with GDC, probably re¯ecting the
con¯icting kinetic properties of NDin and GDC: the
KmNADH of NDin is about 5-fold higher than the KiNADH of
GDC (Douce et al., 2001; Bykova and Mùller, 2001).
Hence, in the absence of complex I and signi®cant extramitochondrial reductant sinks (i.e. in isolated mitochondria
or in leaves in the dark), the accumulation of NADH to a
concentration required to engage NDin substantially is
incompatible with optimal function of GDC.
Intercompartmental redox shuttles during
photosynthesis
The role of mitochondrial matrix enzymes in C3 photosynthesis is well established (Douce and Neuburger, 1989),
and work with Arabidopsis and barley `photorespiratory'
mutants has demonstrated that GDC and serine hydroxymethyl transferase are indispensable for photorespiratory carbon and nitrogen cycling (Somerville and Ogren,
1981; Blackwell et al., 1990). The fate of the matrix
NADH produced by GDC is less clear (Leegood et al.,
1995; Hoefnagel et al., 1998; Foyer and Noctor, 2000;
GardestroÈm et al. 2002). Studies in vitro led to the
conclusion that the reducing equivalents are shared
between the mitochondrial electron transport chain and
exchange with the peroxisome via malate/oxaloacetate
shuttles (Hanning and Heldt, 1993). If this is so in vivo, the
mitochondrial electron transport chain must deal with a
considerable ¯ux of electrons from GDC and a large
fraction of the reductant necessary for peroxisomal
glycerate synthesis must be produced elsewhere, presumably the chloroplast, via the malate valve (Hanning and
Heldt, 1993; Hoefnagel et al., 1998; Scheibe, 2003). When
one considers that the malate valve may also be coupled to
the mitochondrial electron transport chain via cytosolic
and mitochondrial redox shuttles, the picture that emerges
is one of chloroplast and mitochondrial reductant export
co-existing with peroxisomal and mitochondrial reductant
import. Furthermore, in the cytosol, enzymes such as
glyceraldehyde-3-phosphate dehydrogenase may also
make a contribution to NAD(P)H production, whereas
nitrate reductase (NR) acts as an electron sink. According
to this view, the roles of the chloroplast and peroxisome in
redox exchange in illuminated leaves are relatively clear.
Stated simply, the former is a reductant exporter, the latter
51
a reductant importer. Mitochondrial function in the light is
a more complex question. Because the mitochondrial
NADH/NAD ratio is maintained about 100-fold higher
than the cytosolic ratio, KroÈmer and Heldt (1991)
concluded that malate/oxaloacetate exchange must operate
in the direction of net reductant export from the
mitochondria, and that mitochondrial oxidation of
NAD(P)H produced in the cytosol probably occurs through
external dehydrogenases (reviewed by Mùller, 2001).
In the leaves of most C3 species, photosynthesis (and, in
many conditions, the accompanying process of photorespiration) can be very much faster than dark respiration.
The light-dependent photorespiratory pathway complicates the measurement of respiration in the light, though
most data indicate that non-photorespiratory CO2 evolution is signi®cantly inhibited in the light relative to the
dark, whereas inhibition of mitochondrial O2 consumption
is less apparent (Avelange et al., 1991; PaÈrnik and
Keerburg, 1995; reviewed by KroÈmer, 1995; Hoefnagel
et al., 1998). Thus, under many conditions, mitochondrial
electron transport activity in the light is probably maintained, in part, by processes other than `dark' respiration.
What is the predicted effect of photorespiratory Gly
oxidation on total mitochondrial electron transport activity? Given that ¯uxes of carbon into glycollate approach
the rate of net photosynthesis under physiological conditions, it might be considered that when C3 photosynthesis
is rapid, signi®cant partitioning of reductant from GDC
activity to the mitochondrial electron transport chain
would entail much higher respiratory electron transfer
rates than those operating in darkened leaves.
Measurements of respiratory O2 consumption during
active photorespiration are technically dif®cult, notably
because ribulose-1,5-bisphosphate oxygenation and associated glycollate oxidation account for most of the
observed gross O2 uptake (Rey and Peltier, 1989).
Nevertheless, it can be inferred that even during high
rates of photosynthesis in air, a substantial fraction of Gly
oxidation could be coupled to the mitochondrial electron
transport chain without a huge increase in overall electron
transfer rates relative to those that are commonly observed
in darkened leaves (Table 1). This is because (1) only half
the Gly produced must be oxidized, (2) at least part of the
reducing equivalents is exported from the mitochondrion,
and (3) tricarboxylic acid (TCA) cycle activity in the light
is probably signi®cantly less than in the dark (PaÈrnik and
Keerburg, 1995). Nevertheless, the values given in Table 1
suggest that signi®cant coupling of Gly decarboxylation to
the mitochondrial chain would cause some increase in
overall electron transport rates. An attractively simple
mechanism, involving inhibition of pyruvate dehydrogenase by photorespiration-stimulated protein phosphorylation (Gemel and Randall, 1992), has been identi®ed that
could explain how, in the light, mitochondria in photosynthetic cells substitute GDC activity for TCA cycle
52 Noctor et al.
Table 1. Predicted effect of photorespiration on mitochondrial electron transport rates in the photosynthetic cells of C3 leaves
The data compare typical rates of leaf O2 consumption in the dark with maximum and likely rates of mitochondrial O2 consumption at
moderately fast rates of photosynthesis. It is assumed that the ratio of ribulose-1,5-bisphosphate (RuBP) carboxylation:oxygenation is 2.5, which
is a likely value for non-stressed C3 leaves (Keys, 1999); and that `dark' respiration is inhibited 50% by light (see text for references).
Maximum [G] and likely [I] rates of Gly-linked O2 consumption in the light assume that Gly oxidation is coupled either 100% [G] or 40±60%
[I] to the mitochondrial electron transport chain. The basic equation for calculating Gly-linked O2 consumption by the electron transport chain
can be represented by 2 Gly+0.5 O2 ® Ser+CO2+NH3. For purposes of simplicity, O2 consumption that could be linked to export of reductant
from the chloroplast is not considered. For further discussion, see text.
Parameter
Derivation
Value
(mmol m±2 s±1)
[A] `Dark' respiration (in dark)
[B] `Dark' respiration (in light)
[C] RuBP carboxylation
[D] RuBP oxygenation
[E] Glycine oxidation
[F] Net CO2 uptake
[G] Maximum Gly-linked O2 consumption
[H] Maximum total mitochondrial O2 consumption in light
[I] Likely Gly-linked O2 consumption in light
[J] Likely total O2 consumption in light
Typical leaf respiration rate
0.5 [A]
Moderately fast C3 photosynthesis
[C] / 2.5
0.5 [D]
[C] ± ( [B] + [E] )
0.5 [E]
[B] + [G]
0.4 [G] to 0.6 [G]
[B] + [I]
1
0.5
25
10
5
19.5
2.5
3
1 to 1.5
1.5 to 2
turnover. A principal mitochondrial function in the light
could be to generate precursors, notably 2-oxoglutarate (2OG) for net ammonia assimilation (i.e. incorporation of
ammonia other than reassimilation of, for example,
photorespiratory ammonia). This function, which requires
only partial TCA cycle activity, is discussed further below.
Complex I is required for optimal CO2 ®xation
under physiological conditions
Much of the data available on the role of mitochondrial
electron transport in photosynthesis has been generated by
inhibitor treatment of leaf pieces or protoplasts (KroÈmer
et al., 1988, 1993; Igamberdiev et al., 1997, 1998;
Padmasree and Raghavendra, 1999). This and other work
has pointed to several mechanisms by which mitochondrial
electron transport and associated oxidative phosphorylation could be important in optimizing photosynthesis.
First, mitochondrial ATP production could be crucial to
support UDP-glucose formation for sucrose synthesis in
the cytosol. Second, as discussed above, mitochondrial
electron transport is probably responsible for the regeneration of part of the NAD used by GDC. Third, the
mitochondria might act as the ultimate acceptors for
reductant generated in the chloroplast and, therefore,
prevent over-reduction of this organelle.
In the mutant, CMSII, non-function of complex I does
not cause decreased photosynthetic capacity. At saturating
CO2 concentrations, where photorespiration is negligible,
CMSII displays photosynthetic rates at least equal to the
WT (Dutilleul et al., 2003a). Despite the clear restriction
over Gly oxidation in CMSII mitochondria in the dark, in
illuminated leaves the Gly/Ser ratio is not increased
relative to the WT (Dutilleul et al., 2003a). Thus, though
inhibited in the dark, Gly metabolism in CMSII is
supported in the light by components other than complex
I, presumably extra-mitochondrial reductant sinks. As
discussed above, NDin is unlikely to be able to substitute
completely for complex I, though a contribution from
NDin to photorespiratory Gly metabolism cannot be
excluded, given that (1) Gly oxidation is only 70%
inhibited in isolated CMSII mitochondria (Sabar et al.,
2000) and (2) NDin shows some evidence of lightinduction in potato leaves (Svensson and Rasmusson,
2001).
Although light restores Gly metabolism in the mutant,
this comes at a price for the overall photosynthetic process,
suggesting that, in the WT, complex I plays a signi®cant
role in regenerating NAD for GDC activity. Carbon
®xation is consistently decreased in the mutant by 20±
30% in air (Sabar et al., 2000; Dutilleul et al., 2003a).
Analysis of Ci curves and O2 sensitivity demonstrates that
photosynthesis is less inhibited in CMSII, relative to the
WT, under conditions where CO2 ®xation (and, therefore,
sucrose synthesis) is enhanced. This suggests that the
primary cause of decreased photosynthesis in CMSII in air
is unlikely to be insuf®cient mitochondrial ATP production for sucrose synthesis (Dutilleul et al., 2003a). Even
more striking than the decrease in steady-state photosynthesis is the effect on the photosynthetic induction period,
which is greatly prolonged relative to the WT (Dutilleul
et al., 2003a). From these data the following conclusions
can be inferred. First, mitochondrial complex I is largely
dispensable for photosynthesis when photorespiration is
inactive, probably because, in this condition, C3 photosynthesis is limited to chloroplastic events, accompanied
by sucrose synthesis in the cytosol. Second, in air, active
complex I plays an important role in the inter-compartmental redox cycling involved in C3 photosynthesis
(Fig. 2A). Third, when extra-mitochondrial sinks are
Redox interactions in mitochondrial complex I mutants
available in the light, Gly metabolism can continue in the
absence of complex I, but greater demand for other
reductant sinks impacts on redox cycling in the rest of the
cell, decreasing the ef®ciency of the photosynthetic
process (Fig. 2B). This last conclusion is supported by
increased activation state of the chloroplast NADP-malate
dehydrogenase in CMSII, suggesting that the reduction
state of the stroma is increased relative to the WT
(Dutilleul et al., 2003a). One interpretation of the inability
of the mutant to deploy its photosynthetic capacity as
ef®ciently as the WT is that the photosynthetic cell has a
relatively narrow window of redox ¯exibility and that the
mitochondrial electron transport chain plays an important
part in setting the width of this window. Other components
can compensate for the absence of complex I in maintaining Gly oxidation, but this lowers the rate of photosynthesis that can be achieved.
Mitochondria and leaf redox homeostasis:
in¯uence on diurnal regulation and the
determination of stress resistance
The redox changes in CMSII are accompanied by induction or diurnal adjustment of antioxidant systems located
within and outside the mitochondria (Dutilleul et al.,
2003b). Although modulation of antioxidant expression is
usually taken as an indicator of oxidative stress, modi®ed
expression in CMSII occurs despite the absence of any
evidence of increased oxidative load or overall leaf ¯ux to
reactive oxygen species (ROS). In fact, the mutant shows
decreased leaf H2O2 contents, both in the light, where
mitochondrial H2O2 formation is probably a minor
component of total leaf production, and in the dark,
where the mitochondria are probably a major source of
H2O2 (Dutilleul et al., 2003b). Associated with lower leaf
H2O2 is signi®cantly enhanced resistance to both biotic
and abiotic stress, linked at least in part to a higher capacity
of antioxidative enzymes (Dutilleul et al., 2003b). The
increased availability of reductant, as discussed above,
might also play a signi®cant role in a more general upregulation of defence metabolism that underpins observations that CMSII has a higher constitutive resistance than
the WT to a broad spectrum of environmental constraints
and stresses (authors' unpublished results).
As for several other C3 species, there are three genes
encoding catalase in N. sylvestris, and the transcripts of
these genes all vary throughout the day/night cycle.
Whereas CAT1 is most strongly expressed in the dark,
CAT2 and CAT3 transcripts peak in the light (Dutilleul
et al., 2003b). These diurnal changes are enhanced in
CMSII, as is the rhythm for cytosolic ascorbate peroxidase
(cAPX) transcripts, which are highest towards the end of
the photoperiod (Dutilleul et al., 2003b). In contrast to
these transcripts, for which WT expression and/or light/
dark rhythms are reinforced in CMSII, the diurnal pattern
53
of alternative oxidase (AOX) expression is completely
inverted in the mutant. At each point measured throughout
the light/dark cycle, AOX transcripts are more abundant in
CMSII than in the WT. However, AOX transcripts peak in
the light in the WT but are more abundant in the dark in
CMSII, so that the relative increase in AOX transcripts
(CMSII/WT) is about 2-fold in the light but 10-fold or
more in the dark (Dutilleul et al., 2003b). The expression
of AOX has been shown to be inversely correlated with
mitochondrial ROS availability in tobacco (Maxwell et al.,
1999), and AOX capacity can play a role in determining
cell fate in oxidative conditions (Robson and
Vanlerberghe, 2002; Vanlerberghe et al., 2002). While
the factors controlling AOX expression remain fully to be
elucidated, it is possible that mitochondrial redox state,
manifested either as the relative reduction of NAD(P) or of
key electron transport components such as ubiquinone, is
one of the principal determinants of AOX transcript
abundance. Although the intermediaries that link changes
in mitochondrial redox state to nuclear gene expression
remain to be identi®ed, the initial sensing could involve
components such as mitochondrial thioredoxins (Laloi
et al., 2001). Engagement (i.e. in vivo activity) of AOX
protein is dependent on reductive activation, linked to the
reduction status of mitochondrial pyridine nucleotide
pools, probably either through the mitochondrial glutathione system or, more likely perhaps, NADPH-dependent reduction of mitochondrial isoforms of thioredoxin
(Mùller and Rasmusson, 1998; Vanlerberghe and Ordog,
2002). Effects on nuclear transcripts that result from
mitochondrial redox sensing could be multifactorial in
nature, with thioredoxin(s) perceiving the NADPH reduction state and other components such as ROS relaying
information on the status of the electron transport chain
itself.
Mitochondria-linked redox modulation impacts
on the co-ordination of carbon and nitrogen
assimilation
Co-ordination of carbon and nitrogen metabolism in leaves
involves intricate cross-talk at the level of gene expression
and enzyme activity (Stitt et al., 2002; Foyer et al., 2003).
Complementarity and competition also occurs in the use of
metabolites and energy, and the interlocking of C and N
assimilation entails communication between several compartments of the leaf cell, including the chloroplast, the
cytosol, and the mitochondrion (Noctor and Foyer, 1998).
Comparison of foliar metabolite pro®les in CMSII and the
WT indicates that complex I dysfunction impacts strongly
on the leaf C/N interaction (authors' unpublished results).
Thus, even though respiration rates in the dark are not
decreased in the mutant, the relative amounts of metabolites involved in the TCA cycle are modi®ed. Most
strikingly, 2-OG, the carbon skeleton for the glutamine
54 Noctor et al.
Redox interactions in mitochondrial complex I mutants
synthetase/glutamate synthase (GS-GOGAT) pathway
(reviewed by Lea and Mi¯in, 2003), is decreased in the
mutant. By contrast, malate and citrate are generally
increased in CMSII, so that the citrate/2-OG ratio is
typically increased by a factor of 10±20 relative to the WT.
These changes are accompanied by increased ammonia
and Gln/Glu, together with sharp increases in amino acids
with low C/N (Asn, Arg).
Increases in citrate/2-OG, and in Gln/Glu, contrast with
the absence of changes in Gly/Ser in illuminated leaves of
the mutant, and suggest that changes in mitochondrial
redox state impact more strongly on the mitochondria±
chloroplast interactions required for ammonia assimilation
than on GDC activity. Consistently enhanced citrate/2-OG
ratios indicate a restriction over the mitochondrial conversion of citrate to 2-OG, either through modulation of
aconitase or isocitrate dehydrogenase, or both. As the latter
enzyme is NAD-dependent, the simplest explanation of
increased citrate/2-OG in CMSII is that it is a response to
increased reduction of the matrix NAD pool. Thus, the
adjustment in CMSII of the relative pool sizes of TCA
cycle intermediates leads to a perturbation of the GSGOGAT pathway, as shown by an increase in Gln/Glu and
in ammonia. These effects can be predicted to occur when
2-OG supply is low relative to that of ammonia
(Novitskaya et al., 2002). This observation is interesting
given the uncertainty over which isocitrate dehydrogenases (mitochondrial or cytosolic) are most important
in anaplerotic production of 2-OG (reviewed by Hodges
et al., 2003). Theoretical arguments have been advanced in
favour of a predominant role for the cytosolic NADPdependent form (Chen and Gadal, 1990) and indeed
transcripts encoding this enzyme are induced by nitrate
(Scheible et al., 1997). In CMSII, however, the abundance
of this protein is similar to the WT, whereas the
mitochondrial NAD-dependent enzyme is signi®cantly
enhanced (authors' unpublished results). The induction of
the mitochondrial NAD-dependent form indicates that the
de®cit in 2-OG acts, directly or indirectly, as a signal that
speci®cally up-regulates this enzyme. The accompanying
up-regulation of phosphenolpyruvate carboxylase
(authors' unpublished results) is further evidence that
homeostatic activation of anaplerosis occurs in the mutant
in response to 2-OG de®cits, and the consequent perturb-
55
ation of the GS-GOGAT pathway, that result from the
absence of mitochondrial complex I activity.
Complex I de®ciency also causes changes in C/N
interactions downstream of the GS-GOGAT pathway.
Co-ordination of the synthesis of minor amino acids is a
key feature of N metabolism in fungi. This phenomenon,
known as the general control of amino acid synthesis, is
largely unexplored in plants (Foyer et al., 2003). Recent
data suggest the operation of some form of inter-pathway
cross-talk in wheat, potato and barley leaves (Noctor et al.,
2002). The contents of several minor amino acids also
appear to be co-ordinated in N. sylvestris. In the WT,
amino acids with relatively high C/N are most closely coordinated whereas in the mutant, the modulation of the C/
N interaction causes minor amino acid pools to become
dominated by amino acids rich in N (Arg, Lys). Hence, the
perturbation of the GS-GOGAT pathway, involving
decreased 2-OG, increased Gln/Glu, and increased ammonia, feeds downstream to the synthesis of minor amino
acids (authors' unpublished results). This causes a weakening in the co-ordination of minor amino acids, favouring
N-rich amino acids at the expense of C-rich amino acids.
Concluding remarks
The data discussed above demonstrate that mitochondrial
redox state is in¯uential in co-ordinating major leaf
metabolic functions. Redox changes of mitochondrial
origin modulate metabolism and nuclear gene expression.
Key questions concern how such redox changes are sensed.
First, by analogy to the chloroplast plastoquinone pool, the
redox state of an electron transfer component such as
ubiquinone could be important. Second, redox effects on
leaf metabolism and function could be mediated by
changes in pyridine nucleotide pools, which would directly
in¯uence dehydrogenase activities and feed downstream to
in¯uence processes located in other subcellular compartments, such as CO2 ®xation and ammonia assimilation.
Third, effects could occur through modi®ed protein
function and/or gene expression mediated by protein
thiol/disulphide exchange linked to thioredoxins and/or
protein glutathionylation. Thiol-modulation of proteins is a
well characterized mode of post-translational regulation,
particularly in chloroplast metabolism, where thiol/dis-
Fig. 2. Simpli®ed cartoon of the importance of the mitochondrial electron transport chain in redox shuttles during C3 photosynthesis. (A) WT
leaves with optimized photosynthetic activity. (B) One possible explanation of why non-functional complex I renders the Nicotiana sylvestris
mutant, CMSII, less ef®cient for CO2 assimilation under physiological (photorespiratory) conditions (Dutilleul et al., 2003a). It should be
noted that the red bars are meant to indicate restrictions over ¯ux, and not absolute blocks, notably because of the presence of other
components that can at least partly replace complex I function (see text for further discussion). In (B), the proposed sequence of cause±effect
is indicated by numbers. Brie¯y, in spite of the presence of alternative dehydrogenases, the absence of functional complex I (1) limits NADH
oxidation by the mitochondrial electron transport chain (2), which puts extra pressure on reductant export, notably to the peroxisome (3).
This encroaches on a minor, but signi®cant, electron sink for chloroplast reducing equivalents (4), leading to perturbation of redox poise and
decreased photosynthesis (5).
56 Noctor et al.
ulphide exchange is linked to the ¯ux of electrons through
the electron transport chain. In the mitochondria, thioredoxin is reduced by NADPH, which is generated by
enzymes such as NADP-dependent isocitrate dehydrogenase. Finally, modi®ed mitochondrial ROS production
might be one factor responsible for the observed changes
in nuclear gene expression that result from the absence of a
major NADH sink in complex I-de®cient plants.
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