Download A re-evaluation of the ATP :NADPH budget

Journal of Experimental Botany, Vol. 49, No. 329, pp. 1895–1908, December 1998
REVIEW ARTICLE
A re-evaluation of the ATP :NADPH budget during C
3
photosynthesis: a contribution from nitrate assimilation
and its associated respiratory activity?
Graham Noctor1 and Christine H. Foyer
Department of Biochemistry and Physiology, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK
Received 11 July 1998; Accepted 4 August 1998
Abstract
The purpose of this review in reanalysing the
ATP5reductant balance in illuminated leaf cells is to
stress that photosynthesis in vivo does not involve CO
2
fixation alone, but embraces other processes, chief
among which is N assimilation. Prior to the demonstration of CO fixation and photophosphorylation by isol2
ated chloroplasts, the mitochondria were thought
likely to provide all the ATP required for CO fixation
2
(discussed in Arnon et al., 1954). During the 1960s,
the development of techniques for the isolation of
chloroplasts able to fix CO at rates approaching those
2
of the parent tissue induced a paradigm shift, leading
to the establishment of a dominant (if not unanimous)
view that chloroplasts in vivo must by themselves
meet all their ATP requirements. More recent studies,
however, indicate that the reality lies somewhere
between these two extremes. The present work places
emphasis on the integrated nature of photosynthesis
and proposes that much of the respiratory ATP necessary for whole cell photosynthesis may be generated
during the production of C skeletons for N assimilation.
Rather than considering dissipative electron transport
pathways as necessary to uncouple respiratory precursor synthesis from ATP production, the present
analysis emphasizes the metabolic value of ATP produced during N-linked respiration, with cellular ATP
supply being tailored to ATP demand.
Key words: Photosynthesis, ATP :NADPH ratio, CO fixa2
tion, nitrogen assimilation, photorespiration, respiration.
The ATP: reductant requirements of
photosynthetic CO fixation
2
The reductive assimilation of inorganic compounds into
the cellular matter of plants is mediated by low potential
electrons (principally reduced ferredoxin, NADPH,
NADH ) and nucleoside triphosphates (mainly ATP).
These two types of molecule are required in specific
reaction sequences at fixed stoichiometries and their
intracellular pools turn over rapidly (typically, in less
than 1 s). Their relative rates of production must, therefore, be closely co-ordinated with their relative rates of
consumption. Nowhere has this question engendered
more discussion than in the area of photosynthesis where,
since the first demonstration of photophosphorylation
and CO fixation by isolated chloroplasts (Arnon et al.,
2
1954), much attention has focused on the chloroplastic
processes responsible for producing ATP and NADPH
at the stoichiometry required for CO fixation via the
2
RPP pathway (1.5 at the steady-state; Edwards and
Walker, 1983; Horton, 1985). This ratio is increased by
photorespiratory flux and by the consumption of ATP in
the conversion of sugar phosphates to sucrose or starch.
It is still uncertain whether the ATP requirements of C
assimilation can be met solely by linear electron flow to
NADP (for a review, see Horton, 1985). Consequently,
1 To whom correspondence should be addressed. Fax: +44 1582 760981. E-mail: [email protected].
Abbreviations: Ala, alanine; asp, aspartate; 1,3-bPGA, 1,3-bisphosphoglycerate; C:O, ratio of carboxylation:oxygenation of RuBP; FBP, fructose 1,6bisphosphate; Fd, ferredoxin; GAP, glyceraldehyde 3-phosphate; GAPDH, GAP dehydrogenase; gln, glutamine; glu, glutamate; gly, glycine; GOGAT,
glutamine:2-oxoglutarate amidotransferase; GS, glutamine synthetase; HPR, hydroxypyruvate reductase; ICDH, isocitrate dehydrogenase; MDH, malate
dehydrogenase; ME, malic enzyme; NiR, nitrite reductase; NR, nitrate reductase; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; PDH, pyruvate
dehydrogenase; PEP, phospoenolpyruvate; PGA, 3-phosphoglycerate; Pi, phosphate; PK, pyruvate kinase; PSI, photosystem I; PSII, photosystem II;
QR, quantum requirement; Rubisco, RuBP carboxylase/oxygenase; Ru5P, ribulose 5-phosphate; RuBP, ribulose 1,5-bisphosphate; RPP, reductive
pentose phosphate; ser, serine; TCA, tricarboxylic acid; TP, triose phosphate.
© Oxford University Press 1998
1896
Noctor and Foyer
alternative pathways of chloroplastic electron flow, enabling ATP production without NADP reduction, have
frequently been considered necessary. Of these, the most
often considered are cyclic electron flow around PSI
(Arnon et al., 1954; Arnon and Chain, 1975; Heber et al.,
1978; Furbank and Horton, 1987) and O reduction on
2
the reducing side of PSI ( Egneus et al., 1975; Marsho
et al., 1979; Behrens et al., 1982; Furbank and Badger,
1983; Robinson, 1988). Because of the difficulty of directly
measuring the former and the problems of distinguishing
the latter from other routes of O uptake in the leaf, most
2
studies addressing the importance of these mechanisms
have been carried out with in vitro systems. Such studies
have demonstrated the potential of these mechanisms in
supporting light-dependent ATP synthesis in the chloroplast (Arnon et al., 1954; Egneus et al., 1975; Heber
et al., 1978; Furbank and Horton, 1987; Robinson, 1988),
though the actual extent to which they contribute to
ATP synthesis in the intact leaf remains unresolved.
Investigation of relationships between the photochemical
yields of the two photosystems and the quantum yield of
CO fixation have generally failed to provide strong
2
evidence for high rates of either cyclic electron flow or
photosynthetic O reduction in vivo (Genty et al., 1989;
2
Harbinson and Foyer, 1991).
The ATP :reductant requirements of
photosynthetic N assimilation
In the intact plant, photosynthesis cannot be considered
merely as the assimilation of carbon. The second largest
sink for photosynthetic energy in many higher plants is
nitrate. Since the assimilation of nitrate in many species
occurs predominantly in the leaves (Smirnoff and Stewart,
1985), this process will often be ongoing simultaneously
with CO fixation in photosynthetic cells. Elemental ana2
lysis suggests that net rates of N assimilation are around
6–13% of net C assimilation ( Edwards and Walker, 1983;
Raven, 1988; Cram, 1990; de la Torre et al., 1991).
Measured rates show that the capacity for N assimilation
is typically 5–10% of that for CO assimilation
2
( Robinson, 1988). In terms of the required reductant and
ATP, incorporation of nitrate into glu, via NR, NiR, GS,
and GOGAT, can be represented simply by the following
equations:
NO−+NADHNO−+NAD
(1)
3
2
NO−+6Fd NH++6Fd
(2)
2
red
4
ox
Glu+NH++ATPGln+ADP+Pi
(3)
4
Gln+2-OG+2Fd 2Glu+2Fd
(4)
red
ox
In photosynthetic cells, the reaction represented by equation 1 takes place in the cytosol (Smirnoff and Stewart,
1985) but may use reducing power generated in the
chloroplast (Gray and Cresswell, 1984). The reactions
depicted by equations 2–4 occur in the chloroplast stroma,
using photosynthetically reduced Fd (Miflin, 1974; Lea
and Miflin, 1974; Keys et al., 1978). The overall ATP
and reductant balance of equations 1–4 can be represented
as follows:
NO−+2-OG+10e−+ATPglu+ADP+Pi (5)
3
Thus, NO− assimilation into glu requires 2.5 times as
3
many electrons as the reduction of CO to the redox level
2
of TP but only 0.33 times as much ATP. The assimilation
of N is tightly co-ordinated with C assimilation through
a complex array of regulatory processes involving transcriptional and post-transcriptional controls (Foyer et al.,
1995). For example, NO− reduction in leaves is activated
3
by de-phosphorylation of NR, which prevents binding of
the 14-3-3 protein inhibitor (Nussaume et al., 1995;
Bachmann et al., 1996). If plants are deprived of CO ,
2
then N assimilation ceases in both shoots and roots
( Kaiser and Forster, 1989; Pace et al., 1990).
Foliar nitrate assimilation is markedly stimulated by
light (Reed et al., 1983) and can lead to pronounced
increases in light-dependent O evolution (Bloom et al.,
2
1989; de la Torre et al., 1991). The associated photosynthetic electron transport activity is expected to produce
ATP in amounts well in excess of that required for the
reaction catalysed by GS. Thus, as discussed by other
authors ( Edwards and Walker, 1983; Horton, 1985), the
amount of ATP which must be generated during the
production of NADPH for CO fixation is less if nitrate
2
assimilation is also occurring.
The above equations describe simply the reductive
assimilation of NO− into the a-amino group of glu. In
3
order to evaluate the full impact of N assimilation on
cellular ATP status, however, the production of 2-OG
has to be considered. For nitrate assimilation to continue
in the light, 2-OG must be continuously generated, ultimately from photosynthate. This will necessitate at least
partial operation of the glycolytic pathway and the TCA
cycle. It is clear that inorganic N sources stimulate
respiration, increasing the incorporation of newly-fixed
photosynthate into TCA cycle intermediates and amino
acids while diverting carbon from sucrose and starch
(Graham and Chapman, 1979; Larsen et al., 1981; Foyer
et al., 1994; Scheible et al., 1997; Turpin et al., 1997).
Partial operation of glycolytic and TCA cycle activities
to provide carbon skeletons for N assimilation is therefore
another potential source of ATP, in addition to that
generated in the chloroplast during photosynthetic nitrate
reduction.
Dark respiration in the light?
The extent to which respiratory substrate decarboxylations and O uptake occur in illuminated photo2
ATP5NADPH budget during C photosynthesis 1897
3
synthetic cells is still a matter of debate. Using mass
spectrometry to distinguish actual rates of CO uptake
2
and CO release, Avelange et al. (1991) concluded that
2
respiratory decarboxylations were partially inhibited by
light. In contrast, other gas-exchange studies suggested
that the rate of respiratory CO release in the light is
2
equal to, if not higher than, the rate in the dark (AzcónBieto and Osmond, 1983). It seems clear from metabolite
measurements that considerable flux though glycolysis
and the TCA cycle continues in the light (reviewed by
Graham and Chapman, 1979; Krömer, 1995). Indeed,
some oxidative C metabolism must occur, in order to
generate C skeletons for biosynthetic purposes. Significant
amounts of C precursors will be required for processes
such as the shikimate pathway (Hermann, 1995) and
tetrapyrrole synthesis (Castelfranco and Beale, 1983).
Most attention, however, has focused on the role of
respiratory metabolism in meeting the C requirements of
primary N assimilation in the illuminated leaf (Graham
and Chapman, 1979; Larsen et al., 1981; Foyer et al.,
1994; Scheible et al., 1997; Turpin et al., 1997). In
addition to supplying C skeletons, oxidative C flux in
leaves may also be necessary to provide organic acids
important in acid-base regulation during the assimilation
of nitrate in the shoot (Raven, 1988).
If it is now accepted that some respiratory C flow must
occur in the light, in order to sustain biosynthetic pathways, the role of oxidative phosphorylation in the light
is less clear. Data from mass spectrometric analysis suggest that respiratory O uptake is not markedly influenced
2
by light (Gerbaud and André, 1980; Avelange et al.,
1991). However, the mitochondrial inner membrane contains several sites for reductant entry and O reduction,
2
and the ATP yielded during NAD(P)H oxidation can
vary considerably (Azcón-Bieto et al., 1983; Douce and
Neuburger, 1989; Lambers, 1997). Nevertheless, three
studies point to continuing mitochondrial ATP production during photosynthesis. Firstly, it is unlikely that the
cytosolic ATP5ADP ratio in illuminated leaves reaches
the high values required to inhibit oxidative phosphorylation in the mitochondrion (Stitt et al., 1982; Gardeström,
1987). Secondly, respiration can support autotrophic
growth in Chlamydomonas mutants lacking the thylakoid
ATP synthase (Lemaire et al., 1988). Thirdly, perhaps
most importantly, selective inhibition of mitochondrial
ATP production leads to lower rates of photosynthetic
O evolution in leaf protoplasts ( Krömer et al., 1988,
2
1993) and in leaves ( Krömer and Heldt, 1991). This
observation has been rationalized in terms of a requirement for mitochondrial ATP production to support
sucrose synthesis in the cytosol; according to this interpretation, inhibition of mitochondrial ATP synthesis
restricts sucrose synthesis, leading to feedback inhibition
of photosynthesis ( Krömer et al., 1988, 1993).
An integrated approach to cellular ATP5reductant
balance during C photosynthesis
3
Although several authors have considered the influence
of the reductive processes of nitrate and nitrite assimilation on the ATP balance during photosynthesis ( Edwards
and Walker, 1983; Horton, 1985), we are not aware of
any discussion of the impact of the associated, necessary,
oxidative C flow. In this study an integrated analysis of
ATP supply and demand in a leaf cell carrying out
photosynthesis has been undertaken. Photosynthesis will
be considered as the two major reductive assimilations of
inorganic elements (C and N ), together with the synthesis
of carbohydrate and amino acids, photorespiration and
‘dark’ respiration necessary for ongoing N assimilation
in the steady-state. Together, these processes can be
considered as the principal energy-requiring and energyyielding metabolic sequences in the illuminated photosynthetic cell. The calculated data illustrate the potential
importance of N-linked respiratory ATP production in
sustaining high rates of CO fixation. Central to this
2
approach is the recognition that N assimilation in the
illuminated leaf involves two interdependent but distinct
processes: (1) oxidative formation of oxoacids from triose
phosphate, and (2) reductive incorporation of nitrate into
oxoacids to form amino acids.
Underlying assumptions
Analyses of this type must make certain fundamental assumptions, notably concerning the ratio of
ATP5NADPH production by the chloroplast electron
transport chain and coupling factor, which is still a matter
of debate. Here, the value of 1.33 has been chosen as the
starting point for these calculations. This value is derived
assuming that 2 H+ are translocated per electron transported and that 3 H+ (Hangarter and Good, 1982;
Strotmann and Lohse, 1988) are required per ATP synthesized by the chloroplast coupling factor (for further
discussion of these questions, see Edwards and Walker,
1983; Horton, 1985; Ort and Oxborough, 1992; Cramer
et al., 1996). Figure 1 illustrates the metabolic processes
considered in the analysis. Carbon assimilation, photorespiration and NH+ incorporation are assumed to occur
4
via accepted pathways ( Edwards and Walker, 1983; Keys
et al., 1978; Lea and Miflin, 1974; Leegood et al., 1995).
For N assimilation and its associated respiratory activity,
several metabolic alternatives must be considered
( Krömer, 1995). These are indicated on Fig. 1 by numbers, and are described in the legend. It is likely that
these alternative pathways are simultaneously active, to
some degree, under in vivo conditions. However, because
of the difficulty of predicting their relative importance,
various exclusive combinations of these alternatives have
been considered (see Table 2). It should be noted that
only the minimum respiratory activity required for N
1898
Noctor and Foyer
Fig. 1. Diagram of energy production and utilization during the synthesis of carbohydrate and major foliar amino acids in the leaf cell of a C
3
plant. Reaction sequences are not depicted stoichiometrically and not all substrates, co-factors and exchange-transport metabolites are shown. The
processes illustrated are photosynthetic and mitochondrial electron transport, the RPP pathway, the photorespiratory C cycle, starch synthesis,
2
sucrose synthesis, and N assimilation and its associated respiratory C flow. Reaction sequences are shown in detail only for N assimilation. Net
products are denoted by capital letters in boxes. Solid arrows show chemical conversions; dashed arrows indicate translocation between
compartments. For reasons of simplicity, re-incorporation of photorespiratory NH , which is assumed to be complete and to occur via the
3
chloroplastic GS/GOGAT system, is not shown (this process will involve utilization of photosynthetic electrons and ATP and is included in the
calculations of Tables 1 and 2). Unless stated otherwise, the calculations of Tables 1 and 2 assume that hydroxypyruvate reduction is NADHdependent and is perfectly coupled to gly oxidation in the mitochondrion via a malate/OAA shuttle (Hanning and Heldt, 1993; Leegood et al.,
1995). In calculating gas exchange parameters ( Table 1) and cellular ATP balance ( Table 2), it is assumed that the production of oxoacids from
TP occurs by the shortest metabolic route possible, as shown (i.e. the calculations of Tables 1 and 2 consider the minimum oxidation of TP
necessary to support given rates of net amino acid synthesis). Transaminations of pyruvate to ala and OAA to asp are shown to be cytosolic for
reasons of diagrammatic simplicity: these reactions can also occur in the mitochondrion and chloroplast (Ireland, 1997). The data of Tables 1 and
2 are calculated according to different combinations of alternative assumptions about the processes involved in N assimilation. These are marked
by numbers on the figure and are as follows. (1) Reductant for NR activity is provided by glycolytic NADH (1a) formed during conversion of TP
to oxoacids or by export of malate (1b) from the chloroplast (Smirnoff and Stewart, 1985; Scheibe, 1987). (2) TP is oxidized to PGA in the cytosol
by NAD-GAPDH/PGA kinase ( left) or by NADP-GAPDH (right; Kelly and Gibbs, 1973). (3) Pyruvate is produced from PEP via PK (3a) or
via PEPC, NAD-MDH and NAD-ME (3b). (4) Citrate is converted to 2-OG via mitochondrial aconitase and NAD-ICDH (4a) or via cytosolic
aconitase and NADP-ICDH (4b; Chen and Gadal, 1990). For pyruvate production via NAD-ME, it is assumed that the high-affinity mitochondrial
OAA transporter ( Ebbighausen et al., 1985) competes effectively with cytosolic MDH so that OAA is transported into the mitochondrion before
being reduced to malate. Since the two-step conversion of OAA to pyruvate produces and consumes matrix NADH in equal amounts, no ATP
gain will result from import of reducing equivalents into the mitochondria when considering oxidation of cytosolic NAD(P)H by mitochondrial
external dehydrogenases (see Table 2). Abbreviations as in the list, except: NQR, NADH-Q oxidoreductase (complex I ); ext, externally-oriented
NAD(P)H dehydrogenases; bc , cytochrome bc (complex III ), aa , cytochrome c oxidase (complex IV ); FNR, ferredoxin-NADP oxidoreductase;
1
1
3
CF , thylakoid ATP synthase; F , mitochondrial ATP synthase.
1
1
assimilation is considered (partial operation of glycolysis
and the TCA cycle; Fig. 1). Regardless of the route of
pyruvate production, mitochondrial PDH activity would
be required to produce acetyl CoA. Although this activity
is lower in the light than the dark (Budde and Randall,
1990; Gemel and Randall, 1992), it is noteworthy that
only half of the C flowing through PEP is converted to
acetyl units and CO during anapleurotic respiratory
2
activity (Fig. 1).
Through the chloroplastic GS/GOGAT cycle, glu is an
ATP5NADPH budget during C photosynthesis 1899
3
value for C plants under typical atmospheric conditions
3
(Sharkey, 1988; De Veau and Burris, 1989). Values for
two rates of N assimilation are shown that give a ratio
of net C5N assimilation of 19–20.1 (N=5) and 9–10.1
(N=10). These values may be considered conservative
estimates of N assimilation rates in mature and young
leaves, respectively (de la Torre et al., 1991). The %C
incorporated into amino acid pools will vary as a function
of net N assimilation rates and the nature of the amino
acid products ( Table 1). Although the amount of C
incorporated into amino acids may seem substantial, it
should be noted that these values are predicted by likely
in vivo molar ratios of C5N assimilation, at least for the
initial incorporation of N into major foliar amino acid
pools. Early labelling experiments investigating the proportion of fixed C incorporated into total free amino
acids (including ser and gly) gave typical values of 25–40%
after 15–30 min light (Smith et al., 1961; Ongun and
Stocking, 1965). The calculations presented here assume
that, under steady-state conditions, the synthesis of gly
and ser from phosphoglycollate will be matched by their
conversion to sugar phosphates (Fig. 1; Berry et al.,
1978).
Photorespiration increases the required ATP5reductant
ratio in two ways. Firstly, because of lower rates of net
CO fixation per RuBP metabolized, C flow through the
2
early product of N assimilation (Lea and Miflin, 1974).
In the steady-state, transamination reactions will regenerate 2-OG and consume other oxoacids, and amino acids
will be used in further syntheses (for example, proteins,
nucleotides, haem, and chlorophyll ). The complexity of
these reactions forbids a comprehensive assessment of
their influence. Nevertheless, an attempt can be made to
approximate the likely net oxoacid requirements of N
assimilation by considering production of those amino
acids from which the majority of others are synthesized.
Hence, in addition to the synthesis of glu alone, net
production of asp, ala and gln has been considered
( Fig. 1). These compounds, along with photorespiratory
ser and gly, typically represent the major amino acid
pools in illuminated leaves of C plants (Larsen et al.,
3
1981; Noctor et al., 1997). This is reflected in the relative
activities of foliar aminotransferases, of which the highest
(along with GOGAT and the photorespiratory aminotransferases) are glu:pyruvate, glu:OAA, and asp:pyruvate
( Ireland, 1997).
The influence of N assimilation on O and CO exchange
2
2
Table 1 shows the influence of N assimilation and its
associated respiration on gas exchange parameters calculated for given rates of CO fixation (set at 125) and
2
photorespiration (set at 50). A C5O ratio of 2.5 is a likely
Table 1. Effect of N assimilation into different amino acid products on gas exchange parameters
Rate of N assimilation
Amino acid products
0
5
10
—
Glu
Glu
Asp
Ala
Gln
Asp
Ala
Glu
Glu
Asp
Ala
Gln
Asp
Ala
100
—
0
100
0
—
95
19
26
102.5
9.8
1.25
99.5
19.9
21
107
5.1
2.3
100.5
20.1
15.4
108.5
3.2
3.4
90
9
56
105
19
1.25
99
9.9
41
114
9.6
2.3
101
10.1
31
117
6
3.4
Parameter
Net CO fixation
2
C5N ratio (net assimilation)
% Net C fixed into amino acids
Net O evolution
2
Respiratory O uptake (% net O evolution)
2
2
O evolved5O consumed in N assimilation
2
2
Values are in arbitrary units, calculated for absolute rates of RuBP carboxylation and oxygenation of 125 and 50 (C5O=2.5: Sharkey, 1988; De
Veau and Burris, 1989). For operation of the photorespiratory pathway, the calculations assume: (1) all ammonia released from gly is reincorporated
via chloroplastic GS/GOGAT activities; (2) catalase activity results in stoichiometric re-evolution from H O of 50% of the O consumed by
2 2
2
glycollate oxidase activity; (3) gly oxidation is coupled to hydroxypyruvate reduction via redox shuttles, so that the rate of gly-linked respiratory
O uptake is 0; (4) 75% of the C converted to phosphoglycollate is recycled to PGA, and 25% is lost in gly decarboxylation. Thus, for an
2
oxygenation rate of 50, the rate of photorespiratory CO release is 25. Net photorespiratory O consumption is also 25 (50 O consumed at
2
2
2
Rubisco+50 O consumed at glycollate oxidase—25 O released by catalase—50 O evolved at PSII (12.5 linked to GOGAT activity, 37.5 linked
2
2
2
to reduction of 75 1,3-bPGA)). In the absence of N assimilation, therefore, net CO assimilation and net O evolution are both 100 ( left-hand
2
2
column). It is assumed that all net C fixed that is not used for amino acid synthesis is converted to sucrose or starch. N assimilation affects gas
exchange in two ways: (1) O evolution at PSII, linked to the reductive incorporation of NO− into C-amino groups; (2) respiratory CO evolution
2
3
2
and O consumption (Fig. 1). Values on the lowest line represent the rate of photosynthetic O evolution associated with the first process divided
2
2
by the rate of respiratory O consumption associated with the second. The overall C balance for production of the different amino acids can be
2
represented simply as follows: 2TP2-OG+CO (glu and gln); TP+CO OAA (asp); TPpyruvate (ala). For production of glu+asp+ala, and
2
2
gln+asp+ala, relative rates of synthesis are set at 45353 (glu5asp5ala) and 25353 (gln5asp5ala). For the values shown, it is assumed that
photosynthetic electron transport supplies the reductant for conversion of NO− to NO− in the cytosol. The possibility that this conversion utilizes
3
2
NADH generated during respiratory C metabolism is considered in Table 2; coupling of nitrate reduction to respiratory NADH would lead to a
lower rate of respiratory O consumption, but would not change the calculated rates of respiratory CO release, net CO fixation or net O
2
2
2
2
evolution. The above values for gas exchange parameters will be the same for all other alternative pathways and conditions of oxoacid production
considered in Fig. 1 and Table 2.
1900
Noctor and Foyer
regenerative phase of the RPP pathway must be increased
relative to PGA reduction. Thus, for an equivalent rate
of TP production, the ATP5NADPH ratio required in
the RPP pathway increases from 1.5 (no photorespiration)
to approximately 1.54 (C5O=2.5). Secondly, operation
of the photorespiratory pathway introduces an extra ATP
sink at the level of glycerate kinase (Leegood et al., 1995).
This increase in ATP5reductant requirement is slightly
offset by photorespiratory amino acid metabolism and
the necessary re-incorporation of NH ( Keys et al., 1978).
3
Ammonia re-incorporation sustains ATP production
during photosynthetic electron transport to 2-OG
(GOGAT reaction; 1 NADPH equivalent=1.33 ATP
produced per NH re-incorporated ) and utilizes ATP
3
during synthesis of gln (GS reaction; 1 ATP required per
NH re-incorporated ). The net ATP balance will not be
3
affected by possible transamination of glyoxylate by
amino donors other than ser and glu (Betsche, 1983; Ta
et al., 1985). Assuming that gly oxidation and hydroxypyruvate reduction are redox-linked (discussed below),
then the ATP52e− requirement of steady-state incorporation of CO into TP at C5O=2.5 is about 1.57.
2
Where synthesis of several amino acids is considered,
relative rates are set at 45353 (for glu+asp+ala) and
25353 (for gln+asp+ala), ratios which approximate to
the relative pool sizes of these amino acids in leaves from
C plants (see, for example, Noctor et al., 1997). As
3
Table 1 shows, net synthesis of glu alone decreases net
CO fixation by 5%, due to evolution of CO during
2
2
2-OG production. This effect is less marked when synthesis of other amino acids are considered. In fact, net
synthesis of gln, asp and ala leads to a small increase
in net CO fixation, since anapleurotic CO fixation at
2
2
PEP carboxylase exceeds respiratory decarboxylations
( Table 1). Due to its high reductant requirements, N
assimilation markedly increases O evolution, but this
2
effect is offset by respiratory O uptake, so that net O
2
2
evolution is increased only moderately ( Table 1). The
ratio between N-linked O evolution and O consumption
2
2
is lowest for synthesis of glu exclusively ( Table 1, lowest
line), where N assimilation leads to little increase in net
O evolution. When other amino acids are being produced
2
as well as, or instead of glu, the required C flow produces
less NAD(P)H (Fig. 1), potential mitochondrial O
2
uptake is lower and so N assimilation has a bigger effect
on net O evolution ( Table 1). It should be noted that
2
the amount of N-associated O evolution at PSII is
2
independent of the nature of the amino acid end-products,
apart from a slightly lower electron requirement for net
synthesis of gln. The present analysis does not consider
formation of organic acids for acid-base regulation
( Raven, 1988), but is restricted to the production of C
skeletons necessary for ongoing N assimilation. The
assumed rates of oxoacid production required during N
assimilation are therefore conservative estimates.
In photoautotrophic cells at saturating CO , mass
2
spectrometric analysis showed that gross CO fixation
2
was only 75% of gross O evolution (Avelange et al.,
2
1991), indicating reduction of electron acceptors other
than CO . Other acceptors could include nitrate (equa2
tions 1, 2, 4). Indeed, foliar N assimilation in the light
leads to a drop in the assimilatory quotient (net CO
2
fixed5net O evolved; Bloom et al., 1989) and stimulates
2
O evolution (de la Torre et al., 1991). Significantly,
2
however, the observed stimulation of O evolution was
2
less than that predicted from N assimilation rates (de
la Torre et al., 1991). As the values of Table 1 suggest,
this may be explained by concurrent stimulation of photosynthetic O evolution and respiratory O uptake, with
2
2
the latter partially offsetting the former.
Cellular ATP balance during photosynthesis
Table 2 shows a range of values calculated for the cellular
ATP balance when N assimilation and carbohydrate
synthesis are occurring under photorespiratory conditions in the steady-state. Under the assumptions made
for ATP yields of photosynthetic NADP reduction
(ATP5NADPH=1.33), the RPP pathway operating in
isolation (all net CO fixed converted to TP) would show
2
an ATP deficit of 11.1%. In the leaf cell at ambient CO
2
and O concentrations, photorespiration and carbohyd2
rate synthesis will increase the demand for ATP so that,
acccording to our model calculations, the cellular ATP
deficit will be 16.4% and 17.6% for sucrose and starch
synthesis, respectively. As Table 2 shows, even low rates
of N assimilation must remove or at least mitigate this
deficit, due both to the high electron requirement of
reductive N assimilation and to the associated necessary
respiratory C flux (Fig. 1). Important factors affecting
the contribution of N assimilation are the absolute rate,
the nature of the amino acids formed, and the source of
NADH for cytosolic NR activity ( Table 2). The relative
contributions of the reductive and oxidative components
of N assimilation are dependent on these and other
assumptions concerning the route of oxoacid synthesis.
For example, for synthesis of glu, asp and ala at net N=
5 and assuming that photosynthetic reductant drives
nitrate reduction, 33.3 ATP can be formed during production of the necessary 50 low-potential electrons by the
photosynthetic electron transport chain. Under these conditions, a further 43.5 ATP can be generated if the
required 2-OG, OAA and pyruvate (2.051.551.5) are
formed via NAD-GAPDH/PGK kinase and PK, assuming internal oxidation of NADH. In this case, 21% of
fixed C enters amino acid pools (Table 1) and the %ATP
balance is −3.2 or −4.3 (remaining C converted to
sucrose or starch, respectively; Table 2). The total amount
of ATP produced as a result of N assimilation (76.8) is
then 16.5% of that produced by photosynthetic electron
Table 2. The contribution of N assimilation to cellular ATP balance (% deficit or excess)
The calculations use the model pathways and rates given in Fig. 1 and Table 1. Absolute rates of carboxylation and oxgenation of RuBP are set at 125 and 50 (Table 1).
For each set of products at a given rate of N assimilation, the two lines show ATP balance calculated assuming that net C assimilated (Table 1) is converted to the
amino acids shown+sucrose (upper line) or starch ( lower line). In the absence of N assimilation, the %ATP balance is calculated as −16.4 (all assimilated C converted
to sucrose) and −17.6 (all assimilated C converted to starch). Assumptions and conditions additional to those detailed in Fig. 1 and Table 1 are described in the footnote.
Source of reductant for
NRa
Oxidation of TP to PGA
Mitochondrial oxidation
of cytosolic NADHb
Route of pyruvate
production
Amino acid
products
Glu
NAD-GAPDH/PGA kinase
NADP-GAPDH
Internal
Internal
External
External
NAD-GAPDH/PGA kinase
NADP-GAPDH
Internal
Internal
External
ME
PK
External
PK
ME
PK
ME
PK
ME
PK
ME
PK
ME
PK
ME
PK
ME
5
+2.5
+1.5
+21.3
+20.6
+1.6
+0.6
+19.5
+18.8
−0.1
−1.2
+16.0
+15.3
−1.0
−2.1
+14.2
+13.5
+0.7
−0.3
+17.7
+17.0
−0.1
−1.2
+16.0
+15.3
−1.9
−2.9
+12.4
+11.7
−2.8
−3.8
+10.7
+9.9
−1.3
−2.4
+13.6
+12.9
−2.2
−3.2
+11.8
+11.2
−3.1
−4.1
+10.1
+9.4
−4.0
−5.0
+8.3
+7.6
−3.1
−4.1
+10.1
+9.4
−4.0
−5.0
+8.3
+7.6
−5.8
−6.8
+4.7
+4.1
−6.7
−7.7
+3.0
+2.3
5
−3.2
−4.3
+9.8
+8.9
−3.8
−5.0
+8.6
+7.7
−4.8
−5.9
+6.6
+5.7
−5.4
−6.5
+5.4
+4.5
−4.5
−5.6
+7.3
+6.4
−5.1
−6.2
+6.1
+5.2
−6.1
−7.2
+4.2
+3.3
−6.7
−7.8
+2.9
+2.0
−7.1
−8.1
+2.2
+1.3
−7.7
−8.8
+0.9
+0.1
−7.8
−8.9
+0.7
−0.1
−8.4
−9.5
−0.5
−1.4
−6.0
−7.1
+4.3
+3.2
−6.6
−7.7
+3.0
+2.2
−7.6
−8.7
+1.1
+0.2
−8.2
−9.3
−0.1
−1.0
10
5
−6.2
−7.4
+3.9
+2.8
−6.6
−7.8
+3.0
+1.9
−7.3
−8.4
+1.7
+1.1
−7.7
−8.9
+0.9
−0.2
−7.1
−8.3
+2.1
+1.0
−7.5
−8.7
+1.2
+0.2
−8.2
−9.3
0
−1.0
−8.6
−9.8
−0.9
−1.9
−10.0
−11.2
−3.8
−4.8
−10.5
−11.6
−4.7
−5.7
−10.2
−11.4
−4.2
−5.1
−10.7
−11.8
−5.0
−6.0
−7.8
−9.0
+0.5
−0.5
−8.3
−9.5
−0.3
−1.4
−8.9
−10.1
−1.6
−2.6
−9.4
−10.5
−2.6
−3.5
10
Assumptions: reduction of NADP by the photosynthetic electron transport chain is accompanied by the translocation of 4H+ into the intrathylakoid space (for a review, see Horton, 1985).
The H+/ATP requirement of the chloroplast coupling factor is 3 (Hangarter and Good, 1982; Strotmann and Lohse, 1988). The cytosolic NADH/NAD and NADPH/NADP redox couples
are not metabolically linked. Synthesis of the necessary oxoacids for the amino acids shown occurs by the shortest routes possible (Fig. 1). Starch synthesis from TP requires I ATP/hexose
unit (ADP-glucose pyrophosphorylase). Sucrose synthesis from TP requires 1 ATP/sucrose ( UDP-glucose pyrophosphorylase, nucleoside diphosphate kinase). The other ATP sinks are:
chloroplastic PGA (PGA kinase), Ru5P (Ru5P kinase), glycerate (glycerate kinase), glutamate (GS, in photorespiration and net N assimilation). Electron acceptors which sustain chloroplastic
ATP production are: 1,3-bPGA (GAPDH ), 2-OG (GOGAT, in photorespiration and net N assimilation), nitrate (NR, via OAA reduction catalysed by chloroplastic NADP-MDH; see
below), nitrite (NiR).
aPhotosynthetic reductant for nitrate reduction in the cytosol ( left half of table) is provided by a malate/OAA shuttle and so involves no net exchange of ATP between the cytosol and the
chloroplast (Scheibe, 1987). Respiratory carbon flow can provide NADH for NR (right half of table) from NAD-GAPDH, PDH complex or mitochondrial NAD-ICDH activities (it is
assumed that cytosolic NADH from GAPDH is used preferentially). In cases where these activities do not provide sufficient NADH within the present model, the shortfall is assumed to be
made good by export of photosynthetic reductant.
bInternal oxidation of NADH by the mitochondrial electron transport chain has an ADP/O value of 3 whereas this value is 2 for oxidation of cytosolic NAD(P)H by the externallyoriented dehydrogenases of the inner membrane (for a review of ATP yields during operation of glycolysis and the TCA cycle, see Lambers, 1997). In calculating the ATP balance for
‘external’ oxidation of NAD(P)H, 2-OG is assumed to be produced by cytosolic ICDH (Chen and Gadal, 1990): the only dehydrogenase producing matrix NADH is then the PDH complex.
Values for ‘internal’ oxidation of NADH assume full stoichoimetric ‘conversion’ of cytosolic NAD(P)H to matrix NADH by any or all of the following processes: (1) oxidation of isocitrate
inside the mitochondrion rather than in the cytosol; (2) coupling of NAD-GAPD/NADP-GAPDH and NADH-HPR/NADPH-HPR activities (Givan and Kleczkowski, 1992) via redox
shuttles, liberating an equivalent amount of NADH from gly oxidation for oxidative phosphorylation; (3) import of cytosolic NADH into the mitochondrion as malate in exchange for OAA
or aspartate (reversal of the direction of net exchange during coupling of gly oxidation and HPR activity or PDH/NAD-ICDH and NR activities).
ATP5NADPH budget during C photosynthesis 1901
3
Gln
Asp
Ala
Glycolytic/TCA cycle dehydrogenases
Rate of N
assimilation
10
Glu
Asp
Ala
Photosynthetic electron transport chain
1902
Noctor and Foyer
transport associated with C assimilation and photorespiration (466.7), and glycolytic activity and respiration
provide 8% of the total cellular ATP. If the oxoacids are
produced via NADP-GAPDH and ME, and the cytosolic
NAD(P)H formed is externally oxidized by the mitochondria, the ATP generated as a result of oxoacid formation
is only 24 (4.4% of the cell’s ATP) and the calculated
deficit increases to 6.7% or 7.8% (remaining C converted
to sucrose or starch, respectively; Table 2).
When the rate of N assimilation is set at 10 (C5N=
approximately 10; Table 1), the maximum calculated ATP
deficit is 6% ( Table 2; net synthesis of gln, asp and ala,
reduction of nitrate to nitrite by respiratory NADH,
formation of pyruvate via NAD-ME, external oxidation
of cytosolic NAD(P)H ). Other conditions yield a large
ATP surplus (up to 21.3%; net synthesis of glu only,
photosynthetic reduction of nitrate to nitrite, conversion
of TP to PGA via NAD-GAPDH/PGA kinase, production of pyruvate via PK, internal oxidation of NADH by
the mitochondria).
Experimental data suggest that, under certain conditions, nitrate reduction may utilize respiratory NADH,
as shown by nitrate-linked inhibition of respiratory O
2
uptake (Gray and Cresswell, 1984, and references therein).
The extent of this phenomenon in the illuminated leaf
cell is not clear. Coupling of reductant for NR to respiratory C flow has a marked effect on N-linked ATP production (cf. left and right halves of Table 2), since it decreases
both the amount of ATP that can be formed during
photosynthetic nitrate reduction and the amount obtainable by oxidation of respiratory NADH. This effect is
analogous to that resulting from the assumption that the
oxidation of photorespiratory gly is fully coupled to
hydroxypyruvate reduction (Hanning and Heldt, 1993;
Leegood et al., 1995; Raghavendra et al., 1998).
The literature contains conflicting evidence concerning
the fate of mitochondrial NADH during gly oxidation.
Some studies have led to the conclusion that the principal
route of NAD regeneration for ongoing gly oxidation is
likely to be via the mitochondrial electron transport chain
(Dry et al., 1983; Gardeström and Wigge, 1988; Gemel
and Randall, 1992). Other authors have suggested that
some or most of the NADH produced during gly oxidation is shuttled out of the mitochondria as malate (Journet
et al., 1981; Hanning and Heldt, 1993; Raghavendra
et al., 1998).
Despite the theoretically higher ATP5reductant requirements of the photorespiratory cycle, compared to the
RPP pathway, cytosolic ATP/ADP ratios were higher in
barley protoplasts in photorespiratory conditions than at
high CO , suggesting that gly oxidation may be at
2
least partly linked to mitochondrial ATP synthesis
(Gardeström, 1987; Gardeström and Wigge, 1988). If
complete coupling of these reactions was assumed, then
within the confines of the model (oxygenation rate at
Rubisco=50, gly oxidation=25), gly oxidation could
potentially produce as much as 75 ATP in the mitochondria. This would necessitate an alternative source of
reductant for conversion of hydroxypyruvate to glycerate.
If this alternative source were photosynthetic electron
transport, then a further 33.3 ATP could be produced in
driving the reduction of hydroxypyruvate. Hence, complete uncoupling of gly oxidation and hydroxypyruvate
reduction would imply a huge photorespiratory production of ATP, particularly in the mitochondria. In terms
of the total cellular ATP balance, this would produce an
ATP surplus of 3% (sucrose synthesis) or 1.5% (starch
synthesis), even in the absence of N assimilation. This
condition is probably unlikely in vivo (Hanning and
Heldt, 1993; Raghavendra et al., 1998). Nevertheless, it
should be noted that the model calculations of Table 2
do not assume kinetically heterogenous pools of NADH
within the mitochondrion (Dry et al., 1983; Dry and
Wiskich, 1985) and that the coupling of gly oxidation
and hydroxypyruvate reduction is a formal assumption
that merely serves to simplify the calculations. Under
certain combinations of the alternative metabolic routes
considered, it is assumed that cytosolic NAD(P)H produced during respiratory C flow will be coupled to
hydroxypyruvate reduction and that an equivalent
amount of NADH from gly oxidation will be used for
ATP production (Table 2, footnote). The possibility that
hydroxypyruvate serves as an acceptor for photosynthetic
electrons has not been included in the present analysis.
Under some conditions considered in Table 2, the
amount of NADH produced in conversion of TP to
oxoacids would not be sufficient to meet the demands of
nitrate reduction (for example, operation of NADPGAPDH, assuming that foliar NADPH-dependent NR
activity is negligible) and, in these cases, it has been
assumed that the shortfall would be met by photosynthetic
electron transport. Respiration additional to that linked
to N assimilation may also occur in the light and abundant
NADH for nitrate reduction and/or ATP production
might be generated by complete turnover of the TCA
cycle. However, the rate of full cycle turnover in the light
continues to fuel debate. While experiments with labelled
succinate suggest that complete turnover of the TCA
cycle does occur (McCashin et al., 1988), work with
isolated leaf mitochondria metabolizing malate or pyruvate showed that most of the product was exported as
citrate and 2-OG (Hanning and Heldt, 1993). It may be
noted that the present calculations predict that, under
conditions where the ATP balance approaches 0
( Table 2), respiratory O uptake will be 5–10% of net O
2
2
evolution ( Table 1).
Alternative respiratory chain pathways
In calculating the data of Table 2, mitochondrial
NAD(P)H oxidation by internal rotenone-insensitive sites
or the alternative terminal oxidase have not been considered (Douce and Neuburger, 1989). In algae, Weger et al.
(1988) have shown that high rates of NH+ assimilation
4
are associated with a marked increase in cyanide-sensitive
O uptake. Rather than considering dissipative electron
2
transport pathways as necessary to uncouple respiratory
precursor synthesis from ATP production, the present
analysis emphasizes the metabolic value of ATP produced
during N-linked respiration. It is apparent from Table 2
that potential ATP yields derived from oxoacid production vary considerably, and so the efficiency with which
respiratory C flow is linked to ATP generation may be
plastic, with cellular ATP supply being tailored to demand
through, for example, regulation of dehydrogenases by
NADH/NAD ratios and adenylate control of PK.
ATP import into the chloroplast in the light?
The need for mitochondrial ATP production in illuminated protoplasts has been rationalized in terms of ATP
supply to sustain sucrose synthesis in the cytosol ( Krömer
et al., 1988, 1993). However, calculations in this review
suggest that oxoacid generation for N assimilation could
produce significantly more ATP than that required for
the synthesis of sucrose. It should be noted that, although
inhibition of mitochondrial ATP synthesis in illuminated
protoplasts most markedly depressed the cytosolic and
mitochondrial ATP5ADP ratio, a significant effect on the
stromal ratio was also observed ( Krömer and Heldt,
1991). In addition, although the effects of inhibition of
the mitochondrial ATP synthase have been discussed
predominantly in terms of the restriction of sucrose
synthesis, several observations made with oligomycintreated barley leaves may be partly explicable in terms of
inhibition of RuBP regeneration in the RPP pathway due
to decreased ATP supply (eg, lowered RuBP but increased
FBP and TP; Krömer and Heldt, 1991).
Apart from sucrose synthesis, the ATP sinks considered
in the present study are all chloroplastic (Fig. 1). The
mitochondrial electron transport chain could increase
chloroplastic ATP5reductant yields by oxidizing reducing
equivalents exported from the chloroplast (Scheibe, 1987;
Krömer et al., 1988). Since this effect would entail the
mitochondria oxidizing photosynthetically produced
reductant, in addition to reductant produced during respiratory C flow, even higher respiration rates than those
calculated in Table 1 would result.
A direct contribution of mitochondrial ATP synthesis
to chloroplast metabolism would require a means of net
ATP import (Lemaire et al., 1988). Although this could
theoretically be provided by TP import, the net flux of
TP in the light is thought to be from chloroplast to
cytosol (Stitt, 1997). Moreover, in the light, the chloroplastic PGA kinase and GAPDH must catalyse net conversion of PGA to TP in the RPP pathway (Fig. 1).
ATP5NADPH budget during C photosynthesis 1903
3
While these considerations do not rule out an influence
of TP import in supplying ATP to the chloroplast, it
must be recognized that chloroplastic conversion of TP
to PGA would also produce NADPH. Although some
import of TP into the chloroplast may occur, therefore,
the 151 production of ATP and NADPH during its
oxidation to PGA would exacerbate and not mitigate any
shortfall in the required ATP5NADPH ratio.
The chloroplast envelope adenylate carrier (Heldt,
1969) is another putative route through which ATP could
be supplied from the cytosol to the chloroplast, in this
case independently of concomitant transfer of reductant.
The physiological function of this transporter is still a
matter of conjecture, but it has generally been considered
unlikely to play a significant role in photosynthetically
competent leaf cells in the light (Stitt, 1997). One reason
for this is that, in isolated chloroplasts from C plants,
3
maximum rates of the chloroplast ATP translocator are
about 10-fold lower than those of other transporters,
such as the Pi translocator (Flügge and Heldt, 1991). It
is therefore worth considering the predictions of the
present model concerning the relative rates of TP export
from, and ATP import into, the chloroplast. Under
conditions where the cellular ATP balance approaches 0
( Table 2), extrachloroplastic ATP production has a value
of 35–60 and net TP export from the chloroplast to
cytosol is 33.3 (assuming all fixed C goes to amino acids
and sucrose). Subtracting the amount of ATP required
for sucrose synthesis means that respiratory ATP available
for transport to the chloroplast would be 29–54 (about
5–10% of total ATP production by the cell ). Under these
conditions, only 10–17% of the ATP produced extrachloroplastically can be consumed in sucrose synthesis
and the potential net rate of chloroplastic ATP import
would be similar to the net rate of TP export. At high
rates of photosynthesis, therefore, where net fluxes are
likely to be greatest, it would seem improbable that the
chloroplast ATP translocator could keep pace with mitochondrial and cytosolic ATP production, within the confines of the present assumptions concerning the relative
rates of net CO fixation and N-linked respiration.
2
Nevertheless, two points ought perhaps to be borne in
mind. Firstly, unlike the ATP transporter, the Pi translocator is not directional, so that the net flux of TP may be
considerably lower than maximum measured activities
(Stitt, 1997). Secondly, highest activities of the chloroplast
ATP translocator are observed in young leaves (Robinson
and Wiskich, 1977), which have relatively low C5N ratios
(de la Torre et al., 1991).
A flexible view of cellular ATP production
It must be stressed that the model calculations presented
are not intended to provide an exact description of cellular
ATP5reductant balance under all conditions. Clearly, the
1904
Noctor and Foyer
required balance will depend on species and developmental stage, as well as on environmental factors such as
light intensity, temperature and water status. The influence of N assimilation will vary according to the nature
of the external N source and the proportion assimilated
in the leaf.
The sensitivity of photosynthetic CO fixation to inhibi2
tion of mitochondrial ATP synthesis has been shown to
be dependent on ambient conditions ( Krömer et al.,
1993). Other pathways which could potentially function
to adjust the ATP5reductant ratio (for example, cyclic
electron transport, O reduction at PSI ) also occur at
2
variable rates under different conditions, for example,
different light intensities. In spinach chloroplasts fixing
CO , the proportion of electrons allotted to O was
2
2
greater at low light (Heber et al., 1978). Other data
suggest that O reduction (Marsho et al., 1979) and cyclic
2
electron transport (Heber et al., 1978; Furbank and
Horton, 1987) both occur at maximal rates when light
availability is high relative to the capacity of metabolism
to use the products of photochemistry. One such condition, entailing an unusually high demand for ATP, is
during autocatalytic generation of RPP pathway intermediates following a dark–light transition (Marsho et al.,
1979; Furbank and Horton, 1987).
The calculations presented in Table 2 assume that transport of one electron by the photosynthetic electron transport chain results in the transfer of 2 H+ across the
thylakoid membrane. Higher values have sometimes been
observed in isolated thylakoids and are usually explained
in terms of translocation of an extra H+ during oxidation
of plastoquinol at the cytochrome b f complex (Q-cycle;
6
for a review, see Cramer et al., 1996). It remains unclear
whether this phenomenon occurs during intersystem electron transfer under all, or even most, conditions in vivo.
Operation of a Q-cycle may be sensitive to the intrathylakoid pH and, therefore, more likely under conditions
of low membrane energization, for example, low light
( Horton, 1985; Cramer et al., 1996).
On the basis of data obtained in isolated chloroplasts,
it has been suggested that concurrent operation of NADP
reduction and cyclic electron transport will provide more
than enough ATP for CO fixation (Arnon and Chain,
2
1975). Maximum rates of cyclic electron transport require
appropriate redox poising of the electron transport chain
(Arnon and Chain, 1975; Heber et al., 1978). Both cyclic
electron transport and photosynthetic O reduction are
2
inhibited by electron acceptors such as NADP, OAA,
nitrate or nitrite (Heber et al., 1978; Behrens et al., 1982;
Furbank and Badger, 1983; Robinson, 1988). During
reductive N assimilation, therefore, OAA and nitrite are
likely to compete effectively with other pathways of
electron transport. Equally, a contribution of respiratory
ATP production to chloroplast metabolism could also
diminish electron flow from Fd to O or the cyclic
2
pathway, by enabling ongoing oxidation of NADPH in
the RPP pathway. According to this view, the extent to
which electrons engage in alternative pathways would be
flexible and self-regulating, as previously suggested
(Heber et al., 1978).
These calculations of the potential influence of N
assimilation suggest that this process should be taken into
account in considering the ATP balance during C photo3
synthesis, given that, in many photosynthetic cells, C and
N assimilation will be occurring simultaneously. It should
be noted, however, that the range of values shown in
Table 2 emphasize the variable efficiency of ATP production during N assimilation. As in other areas of metabolism, plant cells are likely to show considerable flexibility
in the mechanisms deployed to meet their ATP requirements. Chlororespiration (Garab et al., 1989; Bennoun,
1994), operation of the mitochondrial alternative oxidase
(Azcón-Bieto et al., 1983; Douce and Neuburger, 1989),
and possible increases in proton ‘leakiness’ through CF 0
CF under conditions of high membrane energization
1
(Ort and Oxborough, 1992), are other factors likely to
be sensitive to ATP5reductant status, and therefore able
to contribute in a flexible manner to cellular ATP requirements. The C5O ratio, and the extent to which gly
oxidation and hydroxypyruvate reduction are coupled
during photorespiration (discussed above), are other factors likely to affect, and perhaps be affected by, cytosolic
and stromal ATP5reductant status. Alternative photosynthetic electron transport pathways may be particularly
important in C photosynthesis, where CO fixation
4
2
requires high ATP5NADPH ratios ( Edwards and Walker,
1983). If nitrate reduction does make a significant contribution to the ATP requirements of CO fixation, then
2
this ATP contribution would obviously have to be met
by other processes in plants that assimilate nitrate predominantly in the root. Likewise, in cases where N sources
more reduced than nitrate are assimilated (Raven et al.,
1992), other processes would have to compensate for a
lower ATP yield associated with lower rates of nitrateand/or nitrite-linked photosynthetic electron transport.
The quantum requirements of ATP production associated
with N assimilation
One line of evidence against high rates of photosynthetic
electron flow through pathways other than non-cyclic
electron transport to NADP is the relatively good correspondence found between the measured and expected
quantum requirement (QR) of photosynthesis. The predicted minimum value for the accepted pathways of
electron transport and CO fixation is 8 photons per CO
2
2
fixed (or per O evolved ). On averaging the QR for O
2
2
evolution of 37 different C species photosynthesizing
3
under non-photorespiratory conditions, Björkman and
Demmig (1987) arrived at a value of 9.43. Under the
ATP5NADPH budget during C photosynthesis 1905
3
production associated with the reductive component of
N assimilation would be insufficient to meet the requirements of photosynthesis.
As has been previously noted, the use of photosynthetically-generated reductant in the mitochondria is a
more quantum-efficient way of producing ATP than via
the reduction of alternative electron acceptors in the
chloroplast (Raven, 1976; Krömer et al., 1988). This is
the case whether the reductant is exported from the
chloroplast in the form of malate (Scheibe, 1987) or as
TP, from which reductant is generated during respiratory
oxidation as in Fig. 1. Within the assumptions of the
present calculations, photosynthetic reduction of NADP,
followed by shuttling of reducing equivalents from the
chloroplast to the mitochondrial matrix, could yield a
total of 4.33 ATP per 4 photons or per 2e−. Accordingly,
if both reductive and oxidative components of N assimilation are taken into account, this results in a slightly lower
QR for photosynthesis (15.8) than the minimum calculated assuming that cyclic electron flow or O reduction
2
contributes the required ATP (16.5–17.0; Table 3). It may
be noted that the two conditions for N assimilation which
result in a cellular ATP balance approaching 0 were
chosen since they involve disparate assumptions concerning the rate and routes of N assimilation, and the
products formed. Other combinations of assumptions
would not markedly change this value of 15.8. Equally,
it is noteworthy that none of the calculated minimum QR
values under photorespiratory conditions ( Table 3) are
higher than values that have been determined experimentally for C plants in air (16.7–18.9; calculated from
3
present assumption concerning the amount of ATP
formed during NADP reduction (ratio of 1.33), a QR of
8 is not possible ( Table 3). Eight photons can only
produce 2.67 ATP in reducing 2 NADP via non-cyclic
electron transport. If the ATP deficit is made good by
cyclic electron transport or O reduction, only 1 extra
2
photon will be required and the minimum QR is 9
( Table 3). If reductive N assimilation makes good the
deficit, the minimum QR is approximately 9.2. The slight
increase is due to the relatively small, but nevertheless
significant ATP demands of gln synthesis.
Although the ATP required for sucrose or starch synthesis further increases the calculated minimum QR, the
latter never exceeds 10 in the absence of photorespiration
( Table 3). In contrast, under photorespiratory conditions,
the QR increases drastically, reflecting the potential of
photorespiration as a dissipative protective pathway
( Heber and Krause, 1980). Because the ATP52e− requirements of steady-state CO fixation are increased by photo2
respiration (from 1.5 to about 1.57 for TP production at
C5O=2.5), the proportion of electrons flowing through
the cyclic pathway or to O would have to increase from
2
0.11 to about 0.16 (QR=16.5; Table 3). If the ATP
shortfall is removed purely by reductive N assimilation,
then because of the extra ATP required by the reaction
catalysed by GS, the minimum QR for CO fixation to
2
TP under photorespiratory conditions is slightly higher
(about 16.9: Table 3). Fulfilment of the ATP requirements
of CO fixation purely by reductive N assimilation would,
2
however, entail improbably low C5N ratios (6.8; Table 3)
and so, within the constraints of the present model, ATP
Table 3. Minimum quantum requirements of photosynthesis (quanta per net CO fixed) in the presence or absence of photorespiration
2
and N assimilation
ATP balance
(%)
Additional route of ATP production
Net CO
2
fixation
C5O=2.5
95
TP
Sucrose
Starch
TP
Sucrose
Starch
Sucrose+glu
9.0
9.3
9.5
9.2 (17.2)
9.5 (13.6)
9.8 (11.3)
—
16.5
16.8
17.0
16.9
17.2
17.5
15.8
99
Starch+glu+asp+ala
—
15.8 (9.9)
Cyclic electron transport or photosynthetic
O reduction
2
100
0
Photosynthetic assimilation of nitrate into glu
100
−0.1b
Assimilation of nitrate into amino acids and
associated generation of oxoacids
Assimilation of nitrate into amino acids and
associated generation of oxoacids
Minimum quantum requirement
No photorespiration
0
−0.1a
Products
(6.8)
(6.2)
(5.7)
(19)
Figures in parentheses are net C5N ratios. Calculations assume that 1.33 ATP are generated during non-cyclic reduction of NADP, requiring
absorption of 4 photons. All photosynthetic pathways of electron transport are assumed to produce 0.33 ATP/photon absorbed. Other assumptions
as detailed in Fig. 1 and Tables 1 and 2. The conditions for the values shown in the lower half of the table are taken from Table 2 and are as follows:
aTable 2, row 1, column 6: net rate of N assimilation=5; all N assimilated goes to glu; nitrate is reduced to nitrite using reductant exported from
the chloroplast; TP is oxidized to PGA in the cytosol via NADP-GAPDH; cytosolic NAD(P)H is ‘converted’ into matrix NADH and oxidized by
the mitochondrial complex I; pyruvate is produced via NAD-ME. This condition means that, of the 95C fixed, 25 are used for glu synthesis while
70 are converted to sucrose.
bTable 2, row 8, column 11; net rate of N assimilation=10; N assimilated goes to glu (4N ), asp (3N ) and ala (3N ); nitrate is reduced to nitrite
using reductant produced during TP oxidation; TP is oxidized to PGA in the cytosol via NAD-GAPDH and PGA kinase; cytosolic NAD(P)H is
oxidized by the external mitochondrial dehydrogenases; pyruvate is generated via PK. In this condition, of the 99C fixed, 20 are used for glu
synthesis, 12 for asp synthesis, 9 for ala synthesis, while 58 are incorporated into starch.
1906
Noctor and Foyer
quantum yield values given in Björkman and Demmig,
1987).
Conclusions
A re-evaluation of the ATP5reductant requirements of
photosynthesis has been presented, in which the photosynthetic process is defined as CO fixation, photorespiration,
2
carbohydrate synthesis, N assimilation, and net synthesis
of major foliar amino acids. The calculated data suggest
that ATP production during N assimilation may play an
important role in complementing the relatively high
ATP5reductant requirements of C assimilation. Respiratory C flow necessary for ongoing N assimilation may
contribute significant amounts of ATP in the light, both
through substrate-level and oxidative phosphorylation.
Although alternative electron transport pathways almost
certainly can occur in vivo, these are perhaps best viewed
as facultative rather than obligatory events.
Central to any calculation of photosynthetic
ATP5NADPH balance is the assumed ratio of their
production. This review has used a ratio of 1.33, derived
from H+5e− and H+5ATP ratios of 2 and 3, respectively.
It is possible, by assuming obligatory operation of a
Q-cycle during plastoquinol oxidation at the cytochrome
b f complex (H+52e−=6) and an H+5ATP requirement
6
of 4 (Rumberg et al., 1990), to arrive at an ATP5NADPH
ratio of 1.5, which corresponds exactly to the
ATP5NADPH requirements of CO fixation in the steady2
state (assuming no photorespiration and no starch or
sucrose synthesis). It is hard to see why such a good
correspondence should be expected to exist under all
conditions, given the plethora of other ATP-consuming
and ATP-generating reactions which occur in the illuminated leaf alongside CO fixation. Chief among these
2
reactions will be N assimilation. Other ATP-generating
and ATP-consuming processes, which were not considered
in the present analysis, include sulphate assimilation,
active transport, synthesis of macromolecules, and the
oxidative pentose phosphate pathway. Perfect correspondence of the RPP pathway’s ATP and NADPH
requirements to ATP and NADPH production at the
thylakoid would imply that other ATP-consuming reactions should be exactly matched to cellular ATP generation not linked to photosynthetic NADP reduction. In
contrast to this view, which sees the RPP pathway as
divorced from other cellular processes that produce and
consume ATP, our aim has been to emphasize the integrated nature of photosynthetic metabolism.
Limits over the extent of integration of photosynthetic
metabolism will be set by intracellular compartmentation,
but this is rarely absolute, since transporters enable flux
between compartments. At the present moment, it remains
unclear whether the net flux of ATP into the chloroplast,
either directly or indirectly, would be sufficient to allow
cytosolic and mitochondrial ATP synthesis to contribute
significantly to chloroplast metabolism in the light. It is
suggested, however, that this possibility merits attention,
at least under certain environmental conditions, given the
considerable amounts of ATP that may be formed during
the generation of C skeletons necessary to support likely
rates of N assimilation in the illuminated photosynthetic
leaf cells of C plants. The concept of the chloroplast as
3
a self-sufficient organelle has long been discounted and
there is no reason to suppose that ATP exchange is an
exception. The chloroplast envelope is an active interface
which may be able to match ATP supply and demand on
both cytosolic and chloroplastic sides.
Acknowledgement
This work was prompted by the thought-provoking seminar
given by Ulrich Heber at the University of Paris XI in
November 1997.
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