C S I R O
P U B L I S H I N G
AUSTRALIAN JOURNAL OF
PLANT PHYSIOLOGY
Volume 27, 2000
© CSIRO 2000
An international journal of plant function
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Aust. J. Plant Physiol., 2000, 27, 507–519
Regulation of assimilate partitioning in leaves
Charlotte E. LewisA, Graham NoctorB, David CaustonC and Christine H. FoyerBD
A
School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, UK.
Department of Biochemistry and Physiology, IACR-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, UK.
C
Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, UK.
D
Corresponding author; email: [email protected]
B
This paper originates from a keynote address at the International Conference
on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999
Abstract. Concepts of the regulation of assimilate partitioning in leaves frequently consider only the allocation of
carbon between sucrose and starch synthesis, storage and export. While carbohydrate metabolism accounts for a
large proportion of assimilated carbon, such analyses provide only a restricted view of carbon metabolism and partitioning in leaf cells since photosynthetic carbon fixation provides precursors for all other biosynthetic pathways in
the plant. Most of these precursors are required for biosynthesis of amino acids that form the building blocks for
many compounds in plants. We have used leaf carbon : nitrogen ratios to calculate the allocation of photosynthetic
electrons to the assimilation of nitrogen necessary for amino acid formation, and conclude that this allocation is variable but may be higher than values often quoted in the literature. Respiration is a significant fate of fixed carbon. In
addition to supplying biosynthetic precursors, respiration is required for energy production and may also act, in both
light and dark, to balance cellular energy budgets. We have used growth CO2 concentration and irradiance to modify
source activity in Lolium temulentum in order to explore the interactions between photosynthetic carbon and nitrogen assimilation, assimilate production, respiration and export. It is demonstrated that there is a robust correlation
between source activity and foliar respiration rates. Under some conditions concomitant increases in source activity
and respiration may be necessary to support faster growth. In other conditions, increases in respiration appear to
result from internal homeostatic mechanisms that may be candidate targets for increasing yield.
Introduction
While the relationship between photosynthesis, plant nutrition and agricultural yield is complex, it is certain that future
attempts to enhance yield will increasingly test the limits of
nutritional use and photosynthetic efficiency. Plant dry
matter production is ultimately determined by the balance
between photosynthesis and respiration. A large proportion
of assimilated C is used in maintenance respiration for
upkeep of existing structures and in growth respiration to
produce new components. A substantial part of growth respiration involves oxidation of photosynthate necessary to
produce the organic acid C skeletons required for assimilation of N (Fig. 1). Considerable progress has been made in
the elucidation of metabolic crosstalk between the pathways
of foliar C and N assimilation (Foyer and Ferrario 1994;
Foyer et al. 1994a, b; 2000; Huber et al. 1994; Kaiser and
Huber 1994; Scheible et al. 1997). This research has concentrated on the control of enzyme activity and gene expres-
sion by sugars, nitrate and amino acids. Up to now, no
attempt has been made to refine this comparatively crude
analysis of supply and demand or to model these interactions
in an agricultural context. The yield and quality of harvestable plant parts will depend on appropriate export of C
and N from the source leaf, which will in turn depend on coordinated assimilation rates. Of particular importance to
future agriculture are the predicted increases in ambient CO2
and the consequent effect on source activity (Farrar and
Gunn 1996; Drake et al. 1997).
Nitrogen is frequently limiting in most agricultural
systems and may become more so as a result of global CO2
increases (Smart et al. 1998; Stitt and Krapp 1999). Although
elevated CO2 may sometimes increase absolute N contents,
N often forms a lower proportion of dry weight than in plants
grown in ambient CO2 (Hocking and Meyer 1991). High
crop yields have been achieved with management practices
that rely heavily on N fertilisers. Modelling C:N interactions
Abbreviations used: A, net rate of CO2 assimilation; Asat, light-saturated rate of CO2 assimilation; AOX, alternative oxidase; Bicine, (N, N-bis
[2 -hydroxyethyl]glycine); CABP, 2-carboxy-D-arabinitol-1,5-bisphosphate; Ci , intercellular partial pressure of CO2; DAS, days after sowing; DTT,
dithiothreitol; EDTA, ethylenediamine tetraacetic acid; F ratio, variance ratio distribution; IL, intermediate irradiance (500 µmol m–2 s–1); LL, low
irradiance (150 µmol m–2 s–1); Rubisco, Ribulose-1,5-bisphosphate carboxylase; TCA, tricarboxylic acid; Vcmax, maximum rate of carboxylation.
© CSIRO 2000
10.1071/PP99177
0310-7841/00/060507
508
C. E. Lewis et al.
Metabolism
Growth
Export
CO2
NO3-
Rubisco
NR
Growth
Export
NH4+
SugarP
PEPc
Oxidation
ICDH
Carbohydrates
Organic acids
Amino acids
Fig. 1. Simplified scheme of the distribution of photosynthetically fixed C between synthesis of carbohydrates and oxidative
generation of organic acids for amino acid formation. Ellipses show important regulatory enzymes. NR, nitrate reductase; PEPc,
phosphoenolpyruvate carboxylase; ICDH, isocitrate dehydrogenase.
could be a useful prescriptive approach with which to
achieve enhanced agricultural sustainability at lower N
inputs. At present, low N availability might be expected to
favour dissipatory C loss in respiration as the C:N ratio is
perturbed. Both low N availability and elevated CO2 often
favour carbohydrate accumulation (Rufty et al. 1988; Körner
et al. 1995). Elevated leaf carbohydrate generally favours
enhanced respiration due to higher substrate availability
(Penning de Vries et al. 1979; Azcón-Bieto and Osmond
1983). On the other hand, some reports have shown that
growth at high CO2 leads to decreased respiration (Fock et al.
1979; Gifford et al. 1985) and that elevated CO2 may directly
inhibit succinate dehydrogenase and cytochrome c oxidase
(Azcón-Bieto et al. 1994; Gonzàlez-Meyer et al. 1996).
Other studies have shown that elevated CO2 provokes acclimatory increases in mitochondrial components and numbers
(Robertson et al. 1995; Lewis 1999). In our previous studies
on the C3 grass Lolium temulentum, elevated CO2 stimulated
photosynthesis but produced no increase in biomass (Lewis
et al. 1999). In wheat at elevated CO2, increases in flux
through the AOX were considered a possible explanation of
yield gains that were much smaller than relative increases in
assimilation (Mitchell et al. 1993). While there is limited
literature data indicating increased AOX at high CO2, AOX
activity is known to be dependent on leaf carbohydrate status
(Azcón-Bieto et al. 1983). The recent elucidation of factors
controlling AOX activity suggests that this effect may be
mediated in part through activation by increased concentrations of organic acids, particularly pyruvate and 2-oxoglutarate (Millar et al. 1993; Vanlerberghe et al. 1995), both
of which are important organic acids in N assimilation (Foyer
et al. 2000). In particular, 2-oxoglutarate is the C skeleton for
the primary incorporation of ammonium into amino acids
and is a key player in the co-ordination of C and N assimilation (Ferrario-Méry et al. 2000).
In addition to the necessary supply of C skeletons for N
assimilation and the investment of N in the photosynthetic
apparatus, a fundamental interaction between C and N
assimilation is due to their differing energetic requirements.
We have considered cellular energetics during foliar C and N
assimilation and the necessary interactions between photosynthesis and respiration (Noctor and Foyer 1998, 2000).
Current concepts of regulation are limited by data availability. In particular, we have no experimental knowledge of the
way in which the disparate energetic requirements of C and
N assimilation are reconciled to minimise frequent disruptive oscillations in supply and demand that adversely affect
agricultural yield. The aims of the present work are two-fold:
(1) to use different growth irradiances and CO2 concentrations to modify photosynthetic assimilation and to analyse
effects on respiration at the cellular and whole plant levels;
and (2) to extend our previous modelling approaches to C:N
interactions from the intracellular to the leaf and whole plant
perspectives.
Materials and methods
Plant material and growth conditions
L. temulentum L. (accession number Ba 3081) seeds were germinated
on moist filter paper in closed, plastic transparent boxes, in controlled
environment chambers. Irradiance at the leaf surface was either 150
(shaded plants at low light, LL) or 500 µmol m–2 s–1 (unshaded plants at
intermediate light, IL). The photoperiod (8 h d–1) was kept short to
avoid the initiation of flowering. Day/night temperature was a constant
20°C, and relative humidity 80%. Seedlings were selected for uniformity and vigour 7 d after sowing and transplanted to 2-L seedling
boxes, with a sowing density of 24 plants per box. Plants were grown in
hydroponic culture with an optimal supply of nutrients. The added N
concentration was 1/5th of the original (final concentration 1 mM
NH4NO3), since this was found to be optimal for growth of L. temulentum (Thomas 1983). The nutrient solution was replaced every 2 d in
order to maintain growth and vigour and prevent nutrient deprivation.
Regulation of assimilate partitioning
509
Ninety-six plants were placed in each treatment. They were continuously exposed to ambient (350–360) or elevated (700 µmol CO2 mol–1)
atmospheric CO2 concentrations. Light, temperature and relative
humidity were as for germination. The four growth conditions were:
ambient CO2 at either LL or IL, and elevated CO2 at either LL or IL.
Analyses were performed on fourth leaves 35 d after sowing (DAS), and
experiments were repeated at least three times. By 35 DAS, the fourth
leaves had reached full expansion in all experimental treatments, and
components of the photosynthetic apparatus had fully developed (Gay
and Thomas 1995).
ment as described in Lewis (1999). Only one slice through each cell was
taken and numbers recorded as apparent number per 100 nm section.
Whole plant growth and foliar C:N ratios
A statistical evaluation of the relative effects of irradiance
and CO2 availability on growth and photosynthesis in
Lolium
For each treatment, six plants were randomly selected and harvested
midway through the photoperiod 35 DAS. A further harvest was performed 45 DAS to enable determination of the relative growth rate.
Plants were divided into root, pseudostem, fourth leaf and remaining
leaf blades. Samples were weighed, leaf area was determined using a
Delta-T leaf area meter (Delta-T, Ltd., Cambridge, UK), and material
was dried in a forced-air oven at 80°C. The fourth leaf sub-sample was
finely ground in a hand mill. Samples (2–3 mg) were analysed for N and
C using an isotopic analyser coupled to an Anca-SL preparation module
(Europa Scientific, Crewe, UK).
Leaf photosynthesis
Gas exchange was measured on the fourth leaf using an infrared gas
analysis system (CIRAS, Narrow Leaf Version 1.1-PP Systems,
Hitchin, Hertfordshire, UK). Four plants were selected from each treatment 1 d prior to each harvest date. Leaves were sealed in the leaf
chamber with adaxial surface upwards. Leaves were either measured at
growth conditions (i.e. at growth CO2 and irradiance levels), or at saturating irradiance (940 µmol m–2 s–1) at a range of CO2 concentrations as
described in Lewis et al. (1999). The rate of photosynthetic CO2 uptake
(A) and the intercellular CO2 concentration (Ci) were calculated according to von Caemmerer and Farquhar (1981). The maximum rate of
carboxylation (Vcmax) was estimated from the initial slope of the A/Ci
curves. Constants and temperature corrections at 21 kPa O2 followed
McMurtrie and Wang (1993).
Rubisco activities
Fourth leaves were harvested from four plants per treatment half way
through the photoperiod, immediately frozen in liquid N2, and then
stored at –80°C until analysis. Individual leaves were ground in liquid
N2 and extraction buffer (containing 50 mM Bicine (pH 8.0), 10 mM
MgCl2, 1 mM EDTA and 20 mM DTT) at 2.5 mL g–1 FW. Rubisco activity, activation state and amounts were estimated as described in Lewis
et al. (1999).
Data analysis
Statistical analyses were performed using a two-factor analysis of variance (ANOVA), separating the effects of CO2, light and their interaction. The data for growth were transformed to their natural logarithms
to stabilise variance before the ANOVA application.
Results and discussion
Increasing growth irradiance and CO2 availability were used
to boost source activity in L. temulentum. The light intensities chosen were either close to (500) or just less than half
(150 µmol m–2 s–1) light saturation (Lewis 1999). Statistical
evaluation of the relative effects of irradiance and CO2
enrichment on basic growth parameters is shown in Table 1.
Increasing irradiance had more a significant effect on plant
growth than CO2 availability, and little interaction in plant
response to these variables was observed (Table 1). Some
differential responses were observed (Lewis et al. 1999); for
example, biomass and plant tiller number increased while
leaf area ratio was reduced in response to irradiance. CO2
enrichment also led to an increase in tiller number but this
was observed only at LL, and no significant CO2-induced
effect on biomass or leaf area ratios was evident (Table 1).
Biomass was not affected by CO2 enrichment but was significantly modified by irradiance (Table 1). On average,
biomass was increased by approximately 130% in plants
grown at IL compared to LL (Lewis et al. 1999). Irradianceinduced increases in both above- and below-ground biomass
were observed (Lewis et al. 1999). The ratio of green leaf
area per unit plant weight (leaf area ratio) was significantly
reduced at IL compared to LL. In contrast, CO2 enrichment
had no significant effect on leaf area ratio (Table 1). In
summary, plants grown at LL and high CO2 had a greater
number of shorter tillers than plants growing at the same
irradiance in ambient CO2.
Leaf respiration
Dark respiration rates in fourth leaves were measured at a common CO2
background using a leaf disc oxygen electrode (LD2, Hansatech Ltd,
King’s Lynn, UK). Rates of oxygen uptake were determined at 20°C in
leaf segments harvested at the beginning of the photoperiod. Steadystate rates of respiration were observed within minutes of the onset of
measurement.
Leaf carbohydrates
For each treatment the fourth leaves from four plants were harvested at
0, 4 and 8 h into the photoperiod, and metabolism stopped immediately
by immersion in liquid N2. Soluble sugars and starch were extracted and
measured enzymatically and by HPLC as described by Lewis et al.
(1999).
Microscopy
Segments (2 mm above the tip of the leaf sheath) were harvested at
random from upper and lower surfaces of the leaves grown in each treat-
Table 1. Summary of statistical significance (F ratio) of the effects
of growth CO2 concentration and irradiance on whole plant
biomass, leaf area ratio, and plant tiller numbers in
L. temulentum
Values are F ratios with *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, for
20 d.f. Plants were harvested 35 DAS
Parameter
CO2 concentration
Irradiance
CO2 concentration × irradiance
Mean square
Plant dry
weight
0.56
60.94***
<1
0.065
Leaf area
ratio
Tiller
number
<1
24.44***
<1
0.0037
<1
78.48***
5.43*
0.0037
510
C. E. Lewis et al.
Increasing irradiance had a more significant effect on
photosynthesis than CO2 enrichment, and no significant
interactions between the effects of light and CO2 were found
(Table 2). Photosynthesis was stimulated more by CO2
enrichment at IL than at LL (see below; Lewis et al. 1999).
While IL (500 µmol m–2 s–1) does not approach full sunlight,
this irradiance is likely to be typical within a grassland sward
in temperate zones. Enhanced photosynthetic activity,
whether caused by increased irradiance or by increased CO2
availability, led to similar significant increases in foliar
Rubisco protein contents (Table 2). In contrast to increased
irradiance, CO2 enrichment led to a significant decrease in
the Rubisco activation state (Table 2).
Significant effects of irradiance on foliar carbohydrate
content were observed (Table 2). Leaves from plants grown
at IL contained 232% more sugars (glucose, fructose,
sucrose, trisaccharide and fructans) and 202% more foliar
starch than leaves at LL (Lewis et al. 1999).
Evaluation of energy partitioning between C and N
assimilation from foliar C:N contents: implications for
energy metabolism and whole-plant resource allocation
In plants that assimilate N in the leaves, this process occurs
alongside C fixation, and is a sink for electrons from the
photosynthetic electron transport chain (Fig. 2). The potential influence of light-dependent N assimilation on cellular
adenylate status has been analysed recently (Noctor and
Foyer 1998), but the importance of N assimilation as an electron sink is a matter of some debate. While it is not disputed
that CO2 fixation is the most important electron sink in nonstressed plants under non-photorespiratory conditions
(Genty et al. 1989; Ruuska et al. 2000), the precision limits
of comparative analysis of CO2 uptake and chlorophyll fluorescence probably prevent accurate quantification of lesser
electron sinks, particularly if these operate in proportion to
electrons flowing to C assimilation. For these reasons, the
percentage of electrons accounted for by N assimilation is
often considered to be negligible or low (e.g. assumed to be
not greater than 5%) (Edwards and Baker 1993). Under some
conditions, however, N assimilation may account for a much
bigger proportion of photosynthetic electron flow
(Morcuende et al. 1998).
In theory, this proportion should be derivable from
carbon : nitrogen contents (C:N) of the photosynthetic cell, if
outputs of C and N from the cell can be sufficiently defined.
For a photosynthetic cell in a mature, fully expanded leaf,
these outputs are primarily the cell’s CO2 evolution and its
export of C and N. Therefore:
Cassimilated = C content + respiratory C loss + Cexported
Nassimilated = N content + Nexported
Tables 3 and 4 show attempts to convert literature C:N
values (A) and C:N values we have measured in Lolium (B)
to the fraction of electrons flowing to N assimilation, for a C3
leaf assimilating N from nitrate. Respiratory CO2 evolution
and C export are taken, respectively, as 10% and 50% of
Cassimilated (Wardlaw 1976, 1982; Jeschke and Pate 1991;
Lewis et al. 1999), so that foliar C content = gross C
assimilation (i.e. Cassimilated) × 0.4. For the purposes of calculating assimilation ratios from C:N, and arbitrarily taking N
content = 1:
Cassimilated = C:N × 2.5
Cexported = Cassimilated × 0.5 = C:N × 1.25
To calculate Nassimilated, the value of Nexported must be
known. This parameter is defined in different ways in Tables
3 and 4. In Table 3, phloem loading of C and N is assumed to
occur at a fixed ratio, taken to be Cexported:Nexported = 62.5.
This value is derived assuming that the mean C:N of
exported amino acids is 2.5 and that five sucrose molecules
are exported per amino acid, a figure close to the relative
phloem enrichment of sucrose:amino acid reported from
both measurements of 14C labelling (Madore and Grodzinski
1984) and of total pool sizes (Lohaus et al. 1994 and references therein). For the assumptions of Table 3:
Nassimilated = 1 + (C:N × 1.25/62.5) = 1 + (C:N × 0.02)
Therefore, C:N assimilation rate = C:N × 2.5/[1 + (C:N ×
0.02)]
In this case, where Cexported:Cassimilated and the molar ratio
of Cexported:Nexported are fixed, the proportion of Nassimilated that
is exported (Table 3) will vary according to:
Nexported:Nassimilated = (C:N × 0.02)/[1 + (C:N × 0.02)]
Table 2.
F ratios showing the effects of growth CO2 concentration and irradiance on photosynthetic parameters for
the 4th leaves of L. temulentum at 35 DAS
Values are F ratios with *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, for 12 d.f. (A), 32 d.f. (Rubisco contents) and 44 d.f. (foliar
carbohydrates). Rubisco contents were measured by CABP binding. Data for foliar sugars show summed values for
glucose, fructose, sucrose, trisaccharide and fructan
Parameter
CO2 concentration
Irradiance
CO2 concentration × irradiance
Mean square
A
Rubisco content
7.70*
20.36**
<1
3.47
10.62**
9.42**
<1
0.0814
Rubisco activation state
10.57**
3.16
2.15
51.19
Foliar sugars
Foliar starch
0.34
21.39***
0.028
6.7 × 106
1.12
24.11***
<1
2.7 x 106
Regulation of assimilate partitioning
511
FBP
CHLOROPLAST
Sucrose-P
F6P
UDPG
G6P
CALVIN
CYCLE
CO2
AMINO
ACIDS
amino
acids
PGA
STARCH
NH 4+
SUCROSE
2OG
NO2 -
glucose +
fructose
PEP
pyruvate
NO2-
OAA
glucose +
fructan
Leaf export
VACUOLE
citrate
2OG
CO2
CO2
pyruvate
Roots
SUCROSE
SUCROSE
Leaf export
Roots,
metabolic
sinks
G1P
TP
TP
CO2
NO32OG
CO2
OAA
CO2
acetyl CoA
citrate
TCA
CYCLE
OAA
Roots,
metabolic
sinks
MITOCHONDRION
Fig. 2. Scheme showing relationships between photosynthesis, respiration and the formation of carbohydrates and amino acids. 2-oxoglutarate
(2OG), oxaloacetate (OAA), 3-phosphoglycerate (PGA), triose phosphate (TP), fructose bisphosphate (FBP), glucose 1-phosphate (G1P), glucose
6-phosphate (G6P), fructose 6-phosphate (F6P), uridine diphosphoglucose (UDPG).
For Table 4, it is assumed that the relative rates of C and
N export are governed by their relative rates of assimilation,
so that the relation (Cexported:Cassimilated)/(Nexported:Nassimilated) is
constant. As in Table 3, it is assumed that Cexported:Cassimilated =
0.5, but, instead of a strictly regulated phloem loading of C
and N with Cexported:Nexported set at 62.5, Nexported:Nassimilated is
set at 0.25. This gives (Cexported:Cassimilated)/(Nexported:Nassimilated)
= 2, a value close to data obtained during short-term
labelling experiments of barley leaves (Hanson and Tully
1979). In this case, if other assumptions are the same as for
Table 3:
Cassimilated:Nassimilated = C:N × 2.5/1.25 = C:N × 2
Since the proportions of Cassimilated and Nassimilated that are
exported are now both fixed, the molar ratio Cexported:Nexported
will vary according to C:N × 4 (Table 4).
From the values obtained for C:N assimilated, the proportion of electrons devoted to N assimilation can be calculated
if the electron/N and electron/C values are defined. For both
Tables 3 and 4, these are taken as electron/N = 10 and
electron/C = 7.2 (350 ppm CO2) or 5.5 (700 ppm CO2). The
electron/C values are derived from likely ratios of carboxylation:oxygenation catalysed by Rubisco in unstressed plants
under the two CO2 partial pressures (2.4 and 4.5 at 350 and
700, respectively, Sharkey 1988). The percentage of electrons allotted to nitrate assimilation can be calculated as:
100 × (10/{10 + [C:N assimilation rates × (electron/C)]})
It should be noted that, because electron/C values take
account of photorespiration, the calculations do not require
consideration of photorespiratory CO2 evolution as an output
from the system: CO2 fixation and the photorespiratory
pathway are considered as a single integrated process. The
electron requirements associated with oxygenation are calculated as previously (Noctor and Foyer 1998). ATP generation is assumed to be commensurate with ATP requirements,
regardless of electron partitioning.
The derived data of Tables 3 and 4 suggest that, in plants
growing at optimal N, the proportion of electrons flowing to
N assimilation is of the order of 5–15%. In plants at low N,
512
C. E. Lewis et al.
with very high C:N ratios, the proportion is about 1% (Tables
3 and 4). The relationship between C:N contents and electron
partitioning will be affected by physiological and developmental changes in the rates of various processes, as well as
by differences between species (listed in Table 5).
In excised tobacco leaves supplied simultaneously with
nitrate and sucrose, about 25% of photosynthetic energy was
devoted to N assimilation (Morcuende et al. 1998; Tables 3C
and 4C). If leaf C:N is calculated from this value within the
confines of the model (reverse of procedure for data of
Tables 3 and 4, A and B), then very low values are obtained
(1.6–2.2, Tables 3C and 4C). Clearly, in the case of excised
leaves fed sucrose, our assumptions concerning C and N
export may not apply. Nevertheless, the low C:N values
(which are below those reported in field situations) predicted
from sustained allocation of 25% of electrons to N assimilation suggest that such an allocation is likely to be temporary in attached leaves. Interestingly, however, the data of
Morcuende et al. (1998) were obtained at low light. The data
for Lolium show that foliar C:N during growth at low light is
Table 3.
Predicted relationships between C:N content, C:N assimilation ratios and the proportion of photosynthetic electron transport
linked to nitrate assimilation for 350 and 700 ppm CO2 partial pressure. Relative rates of export are constant
In A, literature data for measured foliar C:N contents (in bold) are converted into mean C:N assimilation ratios by assuming that the photosynthetic
cell respires 10% of the C it assimilates, that the loss of assimilated N is 0, and that C and N are exported from the cell at a molar ratio of 62.5. The
% of electron flow linked to N assimilation is calculated from C:N assimilation ratios by assuming that the number of electrons required is 10 per
N assimilated and 7.2 (350 ppm) and 5.5 (700 ppm) per C assimilated. All electrons are assumed to flow to C assimilation, N assimilation and photorespiration. For details, see text. In B, C:N (the electron allocation to N assimilation) is calculated from data with Lolium (in bold) grown at two different CO2 partial pressures (350 and 700 ppm) and light intensities. In C, literature data for the estimated proportion of electrons linked to N
assimilation (in bold) are converted into C:N assimilation ratios and the foliar C:N contents that would theoretically result from these ratios.
Necessary assumptions as for A
Foliar C:N
contents
Plant
Cassimilated:Nassimilated
Nexported:Nassimilated
% electrons allocated
to N assimilation
Treatment
350
700
350
700
350
700
A Barley
Pea
Tobacco
Tobacco
Grown at optimal N
Grown at optimal N
Grown at optimal N
Grown at low N
8.7A
7.7A
7.0B
72.0B
—
—
—
—
18.6
16.7
15.4
73.8
—
—
—
—
0.148
0.133
0.123
0.590
—
—
—
—
6.9
7.7
8.3
1.9
8.9
9.8
10.6
2.4
B Lolium
Lolium
Grown at optimal N, low light
Grown at optimal N, medium light
4.6C
6.7C
11.8
16.7
10.5
14.8
0.009
0.133
0.084
0.118
0.5
7.7
14.8
10.9
C Tobacco
Tobacco
Tobacco
Excised leaves, fed nitrate + sucrose
Excised leaves, fed nitrate
Excised leaves, fed water
5.2C
7.7C
1.6
4.1
> 13.3
—
—
—
4.2
10.2
> 33.3
—
—
—
0.030
0.081
> 0.268
—
—
—
350
25D
12D
< 4D
700
—
—
—
References for experimental data (in bold): ADe la Torre et al. (1991); BBanks et al. (1999); CLewis et al. (1999); DMorcuende et al. (1998).
Table 4. Predicted relationships between C:N content, C:N assimilation ratios and the proportion of photosynthetic electron transport
linked to nitrate assimilation for 350 and 700 ppm CO2 partial pressure. Relative rates of export are dictated by relative rates of
assimilation
Data, assumptions and data sources as in Table 3, except that N is exported at a fixed proportion of its rate of assimilation (0.25). The relative
export of C and N is therefore described by (Cexported : Cassimilated) / (Nexported : Nassimilated) = 2
Foliar C:N
contents
Plant
Cassimilated:Nassimilated
Nexported:Nassimilated
% electrons accounted
for by N assimilation
Treatment
350
700
350
700
350
700
A Barley
Pea
Tobacco
Tobacco
Grown at optimal N
Grown at optimal N
Grown at optimal N
Grown at low N
8.7A
7.7A
7.0B
72.0B
—
—
—
—
17.4
15.4
14.0
144.0
—
—
—
—
0.348
0.308
0.280
2.880
—
—
—
—
7.4
8.3
9.0
0.9
9.5
10.6
11.5
1.2
B Lolium
Lolium
Grown at optimal N, low light
Grown at optimal N, medium light
5.2C
7.7C
4.6C
6.7C
10.4
15.4
9.2
13.4
0.208
0.308
0.184
0.268
11.8
8.3
16.5
11.9
C Tobacco
Tobacco
Tobacco
Excised leaves, fed nitrate + sucrose
Excised leaves, fed nitrate
Excised leaves, fed water
—
—
—
4.2
10.2
>26.6
—
—
—
—
—
—
25D
12D
< 4D
—
—
—
2.2
5.4
> 17.7
0.008
0.203
> 0.665
350
700
Regulation of assimilate partitioning
Table 5.
2.0
Dark respiration
(µmol O2 m–2 s–1)
A
1.5
35,I
45,I
1.0
45,L
0.5
0
0
4
35,L
8
12
16
20
Net CO2 assimilation ( µmol m–2 s–1)
% increase in photosynthesis :
% increase in respiration
significantly lower than values at high light (Tables 3B and
4B). Values at low light are between 4.6 and 5.2, which yields
a calculated share of photosynthetic electrons of about
10–15%. While not as high as that reported for sucrose-fed
tobacco leaves, these values are still considerable.
The data obtained in Lolium also show interesting effects
of increased CO2 on C:N and energy partitioning between C
and N. In Tables 3A and 4A, measured C:N ratios for barley,
pea and tobacco grown in ambient CO2 are converted into the
proportion of electrons flowing to N assimilation, not only at
ambient but also at 700 ppm CO2. This assumes that modified CO2 availability affects only C:O (i.e. electron/C) and
not C:N. The derived values predict that a higher proportion
of electrons flow to N assimilation as CO2 increases, an
effect purely due to the lesser importance of photorespiration
as an electron sink at higher CO2. In fact, the measured C:N
values for Lolium show that leaf C:N is decreased by elevated CO2, leading to an even bigger increase in the proportion of electrons devoted to N assimilation than predicted,
assuming no effect of growth CO2 on C:N (Tables 3 and 4,
compare A and B).
These analyses assume that light and CO2 availability do
not significantly affect the relationship between net photosynthesis and dark respiration (see discussion below, Fig. 3)
or the proportions of C and N exported. Earlier studies of
short-term effects of light and CO2 in Lolium reported that
Cexported:Cassimilated remained at about 0.4–0.5 over a 5-fold
change in light intensity (Wardlaw 1976) and decreased from
0.59 to 0.46 as CO2 was raised from 320 ppm to 720 ppm
(Wardlaw 1982). Depending on the response of N export to
ambient CO2 availability, this decrease could affect the relationship between C:N and electron allocation to N (Table 5),
although its effect on the derived values of Tables 3 and 4
would be relatively minor.
Comparison of Tables 3 and 4 shows interesting effects of
the assumptions for C and N export on the relationship
513
2
B
1
0
350 ppm
700 ppm
CO2
CO2
Stimulation by growth
at higher light
500 µmol
150 µmol
m-2 s-1
m-2 s-1
Stimulation by growth
at higher CO2
Fig. 3. Relationships between the stimulation of photosynthesis and
respiration by elevated growth CO2 at two different growth irradiances.
(A) Respiration vs photosynthesis at ambient (open symbols) or
700 ppm CO2 (closed symbols) measured 35 or 45 d after sowing at low
(L) or intermediate (I) irradiance. (B) Relative stimulation of photosynthesis and respiration by either growth at higher irradiance (left half)
or higher CO2 (right half) is compared for leaves analysed from plants
35 (open bars) or 45 d (closed bars) after sowing. A value smaller than
1 means that a given growth condition was observed to induce a proportionally greater increase in respiration than in photosynthesis.
Summary of principal factors likely to affect the relationship between leaf C:N contents and the proportion of
electrons flowing to N assimilation in photosynthetic cells
Variable (physiological,
developmental, species-specific, etc)
Assimilation of N sources that are more reduced than nitrate
Use of electrons from respiration for nitrate reduction
Assimilation of N in other parts of the plant
Assimilation of N in the leaf in the dark
Increased contribution of inorganic N to total leaf N
Increase in ratio of C export: N export
Faster rates of alternative electron flow (Mehler reaction)
Decreased rates of respiratory C loss
Decreased partitioning of electrons to photorespiration
Loss of N from the leaf as gaseous N species
Overall leaf C:N ratio is higher than that of the photosynthetic cell
Effect on proportional electron flow to N
assimilation at given leaf C:N contents
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Increase
Increase
Increase
Increase
514
between C:N and electron allocation to N. In plants at optimal N, the assumption that the relation (Cexported:Cassimilated)/
(Nexported:Nassimilated) is constant (Table 4A ,B) predicts a higher
electron allocation to N than that predicted if Cexported :
Nexported is constant (Table 3A, B). When plants are grown at
low N, however, the latter assumption predicts the higher
electron allocation to N (Tables 3A and 4B, data for tobacco).
This is because, for the data of Table 3, Nexported:Nassimilated is
not fixed. Consequently, because the absolute rate of N
export is linked to that of C export, Nexported:Nassimilated attains
a value of 0.59 when C:N is very high (Table 3). This means
that, even though the absolute rate of N assimilation is very
low, the proportion exported increases by a factor or 4 or 5
compared to plants grown at optimal N (Table 3). Such an
effect may be viewed as physiologically advantageous, to
support growth of the plant and thereby mitigate accumulation of C in the leaf. Indeed, the assumption that
Cexported:Nexported is invariant can be seen, in a sense, as regulation of C and N export by sink requirements. When
(Cexported:Cassimilated)/(Nexported:Nassimilated) is constant, then C
and N supply to the rest of the plant will be dictated more by
source capacity. In this case, for tobacco grown at low N, the
assumption that (Cexported:Cassimilated)/(Nexported:Nassimilated) = 2
means that 288 C are exported per N, a 10-fold greater value
for Cexported:Nexported than in tobacco grown at optimal N
(Table 4).
Measurements of phloem C:N in castor bean (grown on
12 mM nitrate) show that ratios depend on leaf age, demonstrating some influence of source activity on relative rates of
C and N export (Jeschke and Pate 1991). The phloem C:N
ratio varied from 19–42, being lowest in young leaves and
highest in older leaves (Jeschke and Pate 1991). These experimental data are within the range of our model values for Nreplete plants. The assumption of Table 3, that C and N are
exported at a fixed ratio of 62.5 irrespective of conditions, is
a conservatively high estimate in N-replete plants. Lower
values would entail an increase in energy partitioning to
nitrate assimilation (Table 5). The two types of control over
C and N export discussed above are obviously not mutually
exclusive, and may complement each other, with their relative contributions perhaps shifting according to environmental variables such as the availability of N.
Source dependency of the balance between photosynthesis,
respiration and export
In Table 6, the amount of C exported in the dark period in
Lolium is calculated by comparing the measured molar
amount of total C stored as carbohydrate in an 8-h light
period with the measured amount of C respired during the
subsequent 16-h dark period. Amino acid contents were not
measured, but, as discussed above, loss of C due to export or
respiration of amino acids will be relatively low. The values
for C respired are derived from rates measured in the dark for
leaf material sampled 1 h into the photoperiod (see
C. E. Lewis et al.
Table 6. The effect of growth CO2 concentration and irradiance
on carbohydrate accumulation, respiration and export
L. temulentum was grown under the conditions described (8-h photoperiod). All measurements were carried out on the 4th leaf. C loss in the
dark period is calculated by subtracting moles of C in total non-structural carbohydrate measured at the end of the dark period from that
measured at the end of the light period. C respired is calculated from
measured respiratory O2 consumption at the end of the dark period and
assuming a respiratory quotient of 1. C exported is calculated as C loss
– C respired. All data are in mmol m–2, except values in parentheses,
which show % of total C loss
Growth condition
[CO2]
(µmol mol–1) Light
350
700
Low
Intermediate
Low
Intermediate
Carbon
loss
Carbon
respired
Carbon
exported
106.4
453.7
118.1
430.4
36.3 (34.1)
77.2 (17.1)
61.1 (51.7)
89.9 (20.9)
70.1 (65.9)
375.5 (82.9)
57.0 (48.3)
340.5 (79.1)
‘Materials and methods’). At this point in the diurnal cycle,
foliar carbohydrate levels were between the low levels measured at the end of the dark period and the higher levels at the
end of the light period. Integrated over the whole dark period,
these respiratory values give an estimate of total dark respiration and enable a comparison of the effects of light and
CO2 on the partitioning of C between export and respiration.
In castor bean, half of the C assimilated photosynthetically by the shoot was translocated to the root
(Jeschke and Pate 1991). In single Lolium source leaves, the
C exported ranged from 48–83%, depending on source activity (Table 6). At low light, CO2 enrichment decreased the
amount of C available for export since the amount of C lost
in respiration was almost doubled (Table 6). With higher
growth irradiance, both at high and low CO2, significantly
more C was exported during the dark period. The absolute
respiratory C loss was higher at higher light levels, but the
proportional respiratory C loss was lower, reflecting the
pronounced increase in C accumulation during the light
period in plants grown at higher irradiance (Table 6).
In L. temulentum, both light and increased CO2 led to
enhanced respiration rates (Lewis et al. 1999). The statistical
significance of these effects is summarised in Table 7, which
also shows that the CO2-dependent decrease in leaf C:N
(Tables 3 and 4) was due to a significant increase in leaf N
(Table 7) of around 10–20% (Lewis et al. 1999). A primary
concern of the present study is the relationship between
photosynthesis and respiration. Figure 3 shows that, when
photosynthesis was increased by higher irradiance or CO2
concentration, respiration was increased in parallel. The relative stimulation of respiration by enhanced CO2 was greater
than that of photosynthesis at low growth irradiance (steep
inclines in Fig. 3A) but this effect was less marked at higher
growth irradiance (shallow inclines in Fig. 3A).
Regulation of assimilate partitioning
515
Table 7. The statistical significance of the effects of growth irradiance and CO2 concentration on foliar respiratory activity and
C:N ratios in L. temulentum
Values are F ratios with *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 for 12 (leaf
respiration) and 20 (leaf N, leaf C:N) d.f. Leaves analysed were from
plants 35 DAS
Parameter
CO2 concentration
Irradiance
CO2 concentration × irradiance
Mean square
Leaf N
C:N
27.05***
223.77***
<1
10.28
30.60***
260.16***
2.48
0.13
Leaf
respiration
10.70*
28.41***
<1
0.054
Developmental stage had relatively little effect on these relationships.
To clarify the relationship between the effect on photosynthesis and respiration when source activity is modified,
the ratio of the stimulation of photosynthesis to that of respiration is plotted in Fig. 3B. At each developmental stage,
respiration was stimulated by a similar factor to photosynthesis. Under some conditions (e.g. effect of high light at
enhanced CO2 45 DAS) divergence from this parity was
evident, but in all cases the ratio of % stimulation of photosynthesis : % stimulation of respiration lay between 0.65 and
1.60. This correlation emphasises the close coupling
between photosynthetic and leaf respiratory capacity.
Associated with the coupled increase in respiration and
photosynthesis caused by such external inputs was an
increase in both chloroplast and mitochondrial numbers
(Table 8). Both elevated CO2 at LL or elevated irradiance in
ambient CO2 increased chloroplast and mitochondrion abundance, suggesting that signals from increased photosynthetic
activity co-ordinate the biogenesis and development of these
energy-transducing organelles. These increases may be
responsible, or at least necessary, for the observed enhancement in photosynthesis and respiration. The effect of higher
light (IL versus LL) could also be partly explained by
increased respiratory substrate concentrations, since higher
growth irradiance produced an overall increase in carbohydrates (Lewis et al. 1999). However, elevated CO2 produced much less significant overall increases in leaf
carbohydrate (Table 2; Lewis et al., 1999), demonstrating
that an increase in respiratory machinery was the principal
cause of faster respiration induced by higher CO2.
The question therefore arises: what are the sensors and
signals that link high ambient CO2 to increased respiration in
Lolium? Although leaf carbohydrate was not generally
increased by higher growth CO2, changes in foliar starch
were evident (Lewis et al. 1999). Our results indicate that upregulation of respiration in Lolium leaves may be linked to
higher starch pools at the end of the night period. A significant regulatory role for the rate of foliar starch degradation
has been proposed by Sun et al. (1999). The utilisation of
sugar phosphates in respiration prevents accumulation of
glucose and sucrose in these circumstances.
It is striking that, although chloroplast and mitochondrion
numbers were greater at either higher irradiance or elevated
CO2 than at low irradiance in ambient CO2, concerted
increases in light and CO2 did not increase numbers further
(Table 8). This may reflect maximal stimulation of organelle
numbers by elevated CO2 or increased irradiance alone.
Although organelle numbers were not increased, enhanced C
and light inputs together did accelerate photosynthesis and
respiration relative to rates observed when only one input
was increased (Fig. 3A, compare I and L, closed symbols). In
this case, the increased flux through the photosynthetic and
respiratory pathways must reflect more enzymatic machinery per organelle, increased substrate availability, or both. It
should be noted that no difference in the overall size of the
chloroplasts was observed (Fig. 4). Examination of the mitochondria, however, showed a marked increase in overall
dimensions at IL. Whereas CO2 enrichment had no effect on
mitochondrial diameter at IL, mesophyll cell mitochondria
were larger than those in ambient CO2 at LL (Fig. 4). Typical
mitochondrial diameters ranged from 1 to 1.5 µm in plants
grown with CO2 enrichment, but were only 700 to 850 nm in
plants grown in ambient CO2 (Fig. 4).
In addition to the effects on chloroplast abundance, irradiance-induced increases in the occurrence of starch grains
in the chloroplast were observed. The number of starch
grains was also increased by CO2 enrichment, but only
during growth at LL. Several starch grains per chloroplast
Table 8.
Apparent numbers of chloroplasts and mitochondria in abaxial and adaxial
mesophyll cell sections of leaves from L. temulentum
Values are in units of apparent numbers per 100 nm leaf section (means ± SE). Leaves analysed
were from plants 35 DAS. Each value is the mean ± SE of 18 sections (9 abaxial, 9 adaxial) taken
from three individual leaves
Growth conditions
[CO2] (µmol mol–1) Irradiance
350
700
350
700
LL
LL
IL
IL
Adaxial
Abaxial
Chloroplasts
Mitochondria
Chloroplasts
Mitochondria
6±0
10 ± 1
11 ± 1
9±1
5±1
8±1
9±0
8±2
6±2
8±1
8±1
9±2
6±1
8±1
8±1
7±0
516
C. E. Lewis et al.
Fig. 4. Electron micrograms of a section of leaf blade grown in ambient (a, b) and elevated CO2 (c, d) and either 150
(a, c) or 500 µmol m–2 s–1 (b, d). Shown are a typical group of adaxial mesophyll cells containing chloroplasts. The bar
represents 5 µm. Mitochondria from individual cells are shown for each treatment. TS shows mitochondria in transverse
sections.
Regulation of assimilate partitioning
were visible under these conditions (Fig. 4), whereas very
few individual grains were observed in chloroplasts of leaves
in ambient CO2 and LL. No marked differences were
observed in the overall size of chloroplasts (data not shown).
Is respiration a target for increasing yield?
The above data illustrate that elevated CO2 does not necessarily lead to improved yield, and that, in Lolium, higher
growth irradiance proved to be a more effective means of
translating increased source activity into increased biomass.
Even when the effects of elevated CO2 were studied during
growth at higher irradiance, where CO2 availability should
be more limiting than at low irradiance, enhancement of
growth CO2 to 700 ppm was unable to stimulate biomass
production. These observations are of considerable potential
relevance to agriculture, in view of the projected increases in
ambient CO2 concentrations within the next century.
Although both higher irradiance and elevated CO2
increased respiration in tandem with photosynthesis, it is
evident that increased respiratory activity only fully counterbalanced enhanced assimilatory capacity when the latter was
stimulated by higher CO2. Accelerated respiration observed
at increased irradiance did not preclude a higher absolute and
proportional allocation of C to leaf carbohydrate and to
export (Table 6). In contrast, elevated CO2 stimulated the
proportion of C that was lost through dark respiration, particularly when the effects of CO2 were studied in Lolium
grown at low light. It is important to consider why light and
CO2 enrichment evoke disparate responses. We may speculate that light elicits a complex repertoire of signal transduction sequences (phytochrome, redox changes, sugars) and
responses, while the message conveyed by CO2 enrichment
is less eloquent because its vocabulary is primarily restricted
to changes in C metabolites.
It should be emphasised that the studies with Lolium were
carried out under short-day conditions, which will tend to
maximise the influence of respiration on leaf C budgets.
Nevertheless, respiration also occurs in the light and so may
impact upon biomass production even during growth at
longer daylengths (Krömer et al. 1993; Krömer 1995).
Moreover, it might be expected that, under short-day conditions where photosynthetic activity is at a premium, any
increase in assimilatory capacity should be exploited to the
full by the plant. Our data suggest, however, that close
coupling between the photosynthetic and respiratory processes may prevent the conversion of increased assimilation
into higher yields. While higher rates of respiration may be
necessary for stimulated growth (e.g. irradiance-induced
increases in respiration were associated with higher growth
rates and biomass production), those brought about by elevated CO2 appeared rather to hinder increases in biomass.
The effect of elevated CO2 may reflect internal homeostatic
mechanisms, perhaps co-ordinated by key C metabolites,
which act to maintain constant relative rates of photo-
517
synthesis and respiration. If so, a significant proportion of
such respiration may be considered dissipatory in nature and
physiologically superfluous.
Since the chief functions of respiratory processes are to
produce C skeletons for biosyntheses and/or ATP for energyrequiring processes, respiration may be dissipatory in one or
both of two senses. Firstly, it may act to burn off excess
carbohydrate as CO2, without production of biosynthetic
precursors (complete TCA cycle activity, Fig. 2). Secondly,
whether producing precursors or not, C oxidation may be
linked to pathways of low ATP yield and therefore uncoupled
to some extent from energy generation. Our previous data
suggest that the increases in respiratory capacity in Lolium
are accompanied by increases in cytochrome c oxidase abundance (Lewis 1999). In addition, accelerated respiration
could also be linked to induction of AOX activity, which may
be necessary to allow high rates of mitochondrial electron
transport under certain conditions (Vanlerberghe et al. 1995;
Igamberdiev et al. 1997).
AOX activity and other pathways of low ATP yield may
also be necessary to confer flexibility in redox state and
adenylate status necessary to avoid potentially damaging disruption of cellular processes during the rapid metabolic flux
that occurs during photosynthesis (Noctor and Foyer 2000).
While changes in key metabolites (pyruvate, citrate)
involved in the regulation of the AOX may contribute to the
overall stimulation of respiration, a more clear-cut effect
appears to be increased abundance of the AOX at both high
CO2 and higher growth light (Lewis 1999). Hence, accelerated respiratory flux in Lolium reflected increases in the
activities of both mitochondrial terminal oxidases. It remains
to be seen whether one viable approach to increasing crop
yields is through targeting components of respiration that
may be ‘unnecessary’ in some environmental and physiological circumstances.
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Manuscript received 28 October 1999, accepted 13 January 2000
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