Plant Physiol. (1998) 118: 573–580
Does a Low Nitrogen Supply Necessarily Lead to Acclimation
of Photosynthesis to Elevated CO2?1
Peter K. Farage, Ian F. McKee, and Steve P. Long*
Department of Biological Sciences, John Tabor Laboratories, University of Essex, Wivenhoe Park, Colchester,
Essex CO4 3SQ, United Kingdom
Long-term exposure of plants to elevated partial pressures of CO2
(pCO2) often depresses photosynthetic capacity. The mechanistic
basis for this photosynthetic acclimation may involve accumulation
of carbohydrate and may be promoted by nutrient limitation. However, our current knowledge is inadequate for making reliable predictions concerning the onset and extent of acclimation. Many
studies have sought to investigate the effects of N supply but the
methodologies used generally do not allow separation of the direct
effects of limited N availability from those caused by a N dilution
effect due to accelerated growth at elevated pCO2. To dissociate
these interactions, wheat (Triticum aestivum L.) was grown hydroponically and N was added in direct proportion to plant growth.
Photosynthesis did not acclimate to elevated pCO2 even when
growth was restricted by a low-N relative addition rate. Ribulose-1,
5-bisphosphate carboxylase/oxygenase activity and quantity were
maintained, there was no evidence for triose phosphate limitation
of photosynthesis, and tissue N content remained within the range
recorded for healthy wheat plants. In contrast, wheat grown in sand
culture with N supplied at a fixed concentration suffered photosynthetic acclimation at elevated pCO2 in a low-N treatment. This
was accompanied by a significant reduction in the quantity of
active ribulose-1, 5-bisphosphate carboxylase/oxygenase and leaf
N content.
Growth at elevated pCO2 frequently brings about change
in plant physiology that is commonly interpreted as acclimation (Drake et al., 1997). Photosynthesis is inextricably
involved because CO2 is the substrate in C3 species that is
limiting at the current atmospheric pCO2. However, results
from investigations on the effects of elevated pCO2 on
photosynthesis have been inconsistent. The stimulatory response brought about when pCO2 is suddenly increased
(Long, 1991) has often been found to decline with increasing duration of exposure (for review, see Gunderson and
Wullschleger, 1994; Sage, 1994; Drake et al., 1997), but some
experiments have failed to find any long-term effect, either
in controlled environments (Radoglou and Jarvis, 1990;
Wong, 1990) or in the field (Arp and Drake, 1991; Jones et
al., 1995; Pinter et al., 1996). Why, then, is the acclimatory
response so varied? Species differences can no doubt account for some of the variability, but often the same species
in apparently similar conditions can yield different results
1
This research was funded by the Biotechnology and Biological
Sciences Research Council (grant no. PG/84/518[W]).
* Corresponding author; e-mail [email protected]; fax 44–1206 –
873416.
with different investigators (Sage, 1994). This fact in itself
suggests that there may be some uncontrolled factor(s) in
the experimental design that may be crucial to the acclimatory response of photosynthesis.
Evidence that additional factors may be interacting with
the CO2 response was brought to prominence by Arp
(1991), who, after reviewing the data from several investigations using a variety of experimental designs, suggested
that root restriction by pot size had a significant effect on
the acclimatory response. Limited rooting volume was suggested to create an imbalance in the supply and demand
for carbohydrates and, consequently, would lead to carbohydrate feedback inhibition of photosynthesis (Stitt, 1991).
However, further investigation has suggested that pot size
and root restriction may only be involved partially in determining the degree of acclimation that occurs at elevated
pCO2; the supply of nutrients is also crucial (Pettersson et
al., 1993). In particular, evidence has accumulated that N
supply is of primary importance. This thesis is particularly
attractive because by far the largest proportion of soluble N
in the leaf is incorporated in Rubisco (Woodrow and Berry,
1988). At elevated pCO2 carboxylation efficiency increases,
enabling the photosynthetic rate to be maintained with less
active Rubisco per unit leaf area. Release of N from excess
Rubisco would then be advantageous if growth was limited by N supply. A number of experimental results now
suggest that acclimation can be significantly slowed by
high-N application (Webber et al., 1994; Drake et al., 1997).
Investigating the role that N supply has on photosynthesis at elevated pCO2 is not easy. When plants are grown in
pots and irrigated with a solution containing a fixed concentration of nutrients, the available N-to-plant mass ratio
will decline with experimental duration. This occurs because there is a finite limit to the quantity of nutrient
solution that can be applied to the pot and also because of
the likely spatial constraint that the roots will progressively
encounter within the container. If elevated pCO2 increases
growth, then the available N-to-plant mass ratio will decline more rapidly, with the danger of confounding the
CO2 treatment with earlier N deficiency (Pettersson and
McDonald, 1994). The RAR method of Ingestad and Lund
(1986) eliminates this problem by supplying N in direct
Abbreviations: Amax, rate of CO2 uptake at light and CO2 saturation; Asat, rate of CO2 uptake at light saturation; ci, intercellular
pCO2; LAR, leaf area ratio; LWR, leaf weight ratio; pCO2, partial
pressure of CO2; RAR, relative addition rate; SLA, specific leaf
area; Vc,max, maximum velocity for carboxylation.
573
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Farage et al.
proportion to the plant growth rate using a hydroponicsculture technique.
To test the hypothesis that acclimation to elevated pCO2
is primarily a response to N availability, wheat (Triticum
aestivum L.) was grown hydroponically at the current atmospheric [CO2] or at 650 mmol mol21 and with either free
access to N or at a relatively low-N RAR. The effects on
photosynthesis were subsequently analyzed and the results
were compared with those of a previous experiment in
which wheat had been grown in sand culture at elevated
pCO2 and the two N treatments were applied in the traditional way by irrigating with solutions containing a fixed
high and low N concentration.
MATERIALS AND METHODS
Plant Material
Winter wheat (Triticum aestivum L. cv Hereward, Plant
Breeding International, Trumpington, UK) was germinated
in 360 or 650 mmol mol21 CO2 on moist filter paper. After
5 d, seedlings of equal size were transferred to hydroponics
troughs.
Hydroponics System
Troughs with sectional covers were used for hydroponically growing the wheat. Each nutrient treatment comprised three troughs, with each trough holding 10 plants.
The nutrient solution was circulated by a centrifugal pump
(model 1060 11 993, Eheim, Deizisau, Germany) to a header
tank, which fed the troughs by gravity, after which the
nutrient solution was collected in a reservoir tank that
resupplied the pump. A diaphragmmatic air pump was
used to ensure that the solutions were continuously aerated. Roots were suspended in the flowing nutrient solution by inserting individual plants into holes in the trough
covers and holding them in place with foam sleeves. The
plant-culture solution was not changed throughout the
course of the experiment and, consequently, care was taken
to choose inert materials that were in contact with the
solution (acrylonitrile butadiene styrene, polyethylene, and
polypropylene). The system was kept scrupulously clean
and light free to minimize growth of microbes and algae.
Nutrient Solutions
The nutrient solutions contained both macronutrients
and micronutrients in the ratio that occurs in healthy wheat
plants (Ingestad and Stoy, 1982). As the plants removed the
nutrients, they were replaced at rates that provided either
free access to all of the nutrients or at a strictly controlled
RAR of N. Thus, the supply of nutrients was continually
increased to match the rising demand of the growing
plants. A detailed description of the principles and techniques for growing plants this way, with a controlled nutrient supply matching the rate of plant growth, has been
described extensively by Ingestad and coworkers (Ingestad
and Stoy, 1982; Ingestad and Lund, 1986). The culture
solution adopted was based on the stock solutions de-
Plant Physiol. Vol. 118, 1998
scribed by Ingestad (1971), adjusted for cereals (Ingestad
and Stoy, 1982) using both nitrate and ammonia as the N
source. The “high-N” treatment provided the plants with
free access to all nutrients and an optimal [N] of 14.3
mm (Ingestad and Stoy, 1982). Nutrients were replenished
in proportion to plant uptake by daily titration with
stock solutions in accordance with conductivity (CL91
Wissenschaftlich-Technische Werkstatten, Weilheim, Germany) and pH (digital pH meter, model CD 620, WPA Ltd.,
Linton, UK) measurements. The same techniques were
used for the “low-N” treatment, except that nitrates were
replaced by chlorides, together with additional adjustments to ensure that the other nutrients remained in correct
proportion in the stock solutions. N (nitrate and ammonia)
was added daily to the low-N treatment at a RAR of 0.07
mol N mol21 N d21. Frequent weighing of the plants
allowed minor corrections to be made in the calculation of
the N required by the plants. Solution conductivities were
kept within 15% of the desired level for the “high-N-free
access” treatment and within 5% of the “low-N-controlled
RAR” treatment.
Sand Culture Experiment
Winter wheat seed was soaked for 24 h before sowing in
washed, lime-free horticultural grit/sand (William Sinclair
Horticultural Ltd., Lincoln, UK) using 0.6-L pots containing drainage holes. Plants were watered as required to
drain through so as to avoid the rooting medium from
drying, using a modified Shive’s solution (Evans and Nason, 1953). The high- and low-N treatments received 10 and
4.5 mm nitrate, respectively. N was added as calcium nitrate and balanced by adding additional calcium sulfate in
the low-N treatment.
Growth Conditions
Plants were grown under artificially lit, controlledenvironment conditions (HPS 1500, Heraeus Vötsch
GmbH, Balingen, Germany). Day and night temperatures
were 20°C/15°C, the water vapor pressure deficit was ,0.7
kPa, and the photoperiod was 14 h, with a PPFD at leaf
height of approximately 750 mmol m22 s21. The [CO2] was
controlled at 360 or 650 6 15 mmol mol21 CO2 using a
combined IR gas analyzer and microprocessor unit (model
WMA-2, PP Systems, Hitchin, UK). CO2 was supplied from
a compressed gas cylinder (Linde Gas UK Ltd., Stoke on
Trent, UK) certified as 999.95 mmol mol21 CO2, ,0.5 mmol
mol21 C2H4. Before entering the controlled environment
chamber the gas was passed through a potassium permanganate column as a further precaution against contamination by hydrocarbons.
Wet Weight, Dry Weight, and Leaf-Area Measurement
To ensure that plant growth in the hydroponics experiment was increasing in accordance with the rate of N
addition in the low-N treatment and that growth of the
control plants was uninhibited, the fresh weight of 10
plants was measured every 2 to 3 d. During this procedure
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Photosynthetic Acclimation and Nitrogen Supply
the roots were kept submerged, only removed from solution for the short time it took to dab off surplus water with
absorbent paper and weighed on a top pan balance (model
HC22, Oertling, Smethwick, UK).
At the end of the growing period (ligule emergence of
the sixth leaf) plants were divided into their component
parts (roots, pseudo stems, leaf laminae, and tillers). Leaf
area was measured with a leaf-area meter (Delta T Devices,
Burwell, UK). The plant tissue was dried to constant mass
in a fan-assisted oven at 80°C before weighing on an analytical balance (model 2006 MP, Sartorius, Göttingen, Germany), which was self-calibrating and cross-checked annually by the manufacturer.
Gas-Exchange Measurements
Leaf gas-exchange measurements were made using a
portable IR gas analysis system (CIRAS-1, PP Systems) and
a narrow leaf cuvette with a quartz-iodide light source
(PLC, PP Systems). The CO2 and water analyzers were
routinely calibrated against a CO2 standard (Linde Gas UK
Ltd.) and water vapor generator (model WG600, ADC Ltd.,
Hoddesdon, UK). The PPFD at the level of the leaf was
1400 mmol m22 s21, whereas the rest of the plant remained
at the controlled-environment growth conditions. Responses of photosynthetic CO2 uptake to changes in pCO2
over the range of 50 to 150 mmol mol21 and 1200 to 1600
mmol mol21, at pO2 of 210 mmol mol21, were made to
calculate the Vc,max and the Amax. Calculation of Vc,max
followed the procedure of McKee et al. (1995). The effect of
inhibiting photorespiration was investigated by reducing
the pO2 from 210 to 21 mmol mol21.
Tissue Analysis of Rubisco and N
Samples for the Rubisco assays were taken from the
central portion of the sixth leaf at ligule emergence, i.e.
575
identical sections to those used for gas-exchange measurements. The sections were collected halfway through the
photoperiod and immediately immersed in liquid N2. Extraction and assay of Rubisco activity, activation, and content were as described in McKee et al. (1995).
Total leaf N content was measured by GC using an
elemental analyzer (model PE 2400 series II CHNS/O
Analyser, Perkin-Elmer Cetus). Samples were first ground
to a fine powder and the instrument was calibrated with
acetanilide standards (Perkin-Elmer Cetus).
Statistical Analysis
The data were analyzed using two-way analysis of variance (Systat Inc., Evanston, IL) with pCO2 and N as independent factors. Post-hoc pairwise comparisons were made
using Scheffé’s probability. Growth rates of the hydroponically grown plants were transformed and analyzed by a
regressions comparison (Sokal and Rholf, 1995).
RESULTS
The two N treatments used in the hydroponics-culture
technique produced wheat plants with significantly different rates of growth (P . 0.01), but pCO2 had no significant
effect on growth rate (P . 0.05; Fig. 1a). The elevatedpCO2-grown plants did, however, exhibit an initial slight
growth advantage and so were always bigger than their
ambient-pCO2-grown counterparts. Growth was exponential at 0.18 and 0.20 d21 for the control and elevated-pCO2grown plants with free access to N and was 0.09 and 0.10
d21 for the control and elevated-pCO2 plants grown at the
restricted rate of N supply (Fig. 1b). There was no significant effect of CO2 treatment (F 5 1.609, P ,0.2) or N supply
(F 5 0.021, P ,0.8) on Asat, so there was no evidence that
photosynthesis was down-regulated by elevated pCO2
Figure 1. The effects of pCO2 and N supply on the increase in total wet weight (a) and ln wet weight (b) of wheat. Plants
were grown hydroponically with day/night temperatures of 20°C/15°C and a photosynthetically active photon flux density
at leaf height of approximately 750 mmol m22 s21. Treatments were: f, 650 mmol mol21 CO2, free access to N; M, 360 mmol
mol21 CO2, free access to N; F, 650 mmol mol21 CO2, RAR of 0.07 mol N mol21 N d21; and E, 360 mmol mol21 CO2,
RAR of 0.07 mol N mol21 N d21. Vertical bars represent SE; n 5 10.
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576
Farage et al.
Figure 2. Rates of Asat and Vc,max of the sixth leaf at ligule emergence
of wheat grown hydroponically (hy), and the Vc,max of the sixth leaf
at ligule emergence of wheat grown in sand culture (sd). Measurements were made with a leaf temperature of 23°C with a photosynthetically active photon flux density of 1400 mmol m22 s21 and pO2
of 210 mmol mol21. The Asat was measured at 360 mmol mol21 CO2,
and Vc,max was obtained from the initial slope of the CO2 response
curve. Treatments consisted of the following: Hydroponics: f, 650
mmol mol21 CO2, free access to N; M, 360 mmol mol21 CO2, free
access to N; s, 650 mmol mol21 CO2, N RAR of 0.07 mol N mol21
N d21; o, 360 mmol mol21 CO2, N RAR of 0.07 mol N mol21 N d21.
Sand culture: f, 650 mmol mol21 CO2, 10 mmol nitrate; M, 360
mmol mol21 CO2, 10 mmol nitrate; s, 650 mmol mol21 CO2, 4.5
mmol nitrate; o, 360 mmol mol21 CO2, 4.5 mmol nitrate. Vertical
bars represent SE; n 5 4.
(Fig. 2). An identical pattern of results was obtained when
the measurements were made at 650 mmol mol21 CO2 (data
not shown), which is commensurate with this apparent
lack of acclimation. The Asat of the elevated pCO2-grown
plants measured at their growth pCO2 at the sixth-leaf
stage was significantly higher (56%) than the rate obtained
for plants grown and measured at 360 mmol mol21 (P
,0.01).
Plant Physiol. Vol. 118, 1998
Analysis of tissue N showed that the low-N hydroponically grown plants exhibited a significant reduction (P
,0.001) in the N content of each plant organ (roots, stems,
and leaves) compared with controls, although the leaves
suffered the smallest decrease (Table I). This change in N
content was accompanied by a significant increase in the
C-to-N ratio of each organ (Table I). However, when N
content was expressed on a leaf-area basis, the effect of N
treatment was greatly reduced at the sixth-leaf stage and
was only significantly different between N treatments of
the elevated pCO2 leaves (Table I; F 5 14.037; P . 0.01).
This result is apparently due to the LAR, which showed the
greatest change in response to CO2 treatment (Table II),
brought about by a decrease in SLA (P ,0.001) rather than
a decrease in LWR (P 5 0.1).
A comparison with results from the sand-culture experiment shows that leaf N content on a leaf-weight basis was
not substantially lower than that of the hydroponically
grown plants, but only when plants were irrigated with the
high-N treatment (Table III). Those plants given the low-N
solution suffered a large, significant reduction in leaf N
content even when expressed on a leaf-area basis (Table III;
F 5 130.503; P . 0.001). Elevated pCO2 exacerbated this
reduction in [N] (F 5 13.650; P . 0.001). This result occurred in spite of the low-N sand-culture plants receiving a
higher total N dose over the course of the experiment than
their hydroponically grown counterparts. Thus, growing
wheat with fixed [N] dramatically reduced leaf N content;
an effect that was augmented by elevated pCO2, whereas
growth in hydroponic culture had very little effect on leaf N.
The failure of elevated CO2 to bring about a reduction
of N content in hydroponically grown wheat may be a
crucial factor in the ability of these plants to avoid photosynthetic acclimation as determined by Asat. Central to
this effect is likely to be the response of Rubisco. Figure 3
shows A/ci curves for the sixth leaves once they had
reached full expansion. Plants grown and measured at the
current ambient pCO2 at high and low rates of N supply
had values of ci on the initial phase of the curve, inferring
Rubisco limitation. Growth at elevated pCO2 shifted the
operating point to the inflection of the curve (Fig. 3),
Table I. Tissue N and C-to-N ratio of hydroponically grown wheat
Tissue N and C-to-N ratio of hydroponically grown wheat in 360 mmol mol21 CO2 (LC) or 650 mmol mol21 CO2 (HC) with either free access
to N (HN) or at a low rate of N supply (LN). Plants were harvested when the sixth leaf had fully expanded. Values shown are the means 6 SE
per unit dry weight; n 5 4 to 5.
Plant Nitrogen
Tissue N concentration
All leaves (mg g21)
Leaf 6 (g m22)
Shoot (mg g21)
Root (mg g21)
C-to-N ratio
Leaf
Shoot
Root
a
ANOVAa
Treatment
LCHN
HCHN
LCLN
HCLN
66.1 6 0.7
2.7 6 0.1
59.1 6 1.1
50.1 6 1.4
60.3 6 3.9
3.0 6 0.1
54.4 6 2.6
48.2 6 0.6
55.6 6 0.7
2.3 6 0.21
39.3 6 1.6
35.7 6 1.2
52.2 6 0.7
2.0 6 0.4
39.7 6 4.1
36.6 6 1.3
6.4 6 0.1
6.9 6 0.1
8.0 6 0.0
6.9 6 0.4
7.2 6 0.2
7.8 6 0.2
8.1 6 0.1
10.6 6 0.3
11.7 6 0.4
8.5 6 0.1
10.6 6 1.0
11.6 6 0.3
CO2
N
***
**
***
***
*
***
***
***
ANOVA, Analysis of variance: *, P , 0.05; **, P , 0.01; ***, P , 0.001. There were no significant interactions between CO2 and N.
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Photosynthetic Acclimation and Nitrogen Supply
577
Table II. Leaf growth analysis of hydroponically grown wheat
LAR, SLA, and LWR of hydroponically grown wheat in 360 mmol mol21 CO2 (LC) or 650 mmol mol21 CO2 (HC) with either free access to N (HN)
or at a low rate of N supply (LN). Plants were harvested when the sixth leaf had fully expanded. Values are the means 6 SE.
Leaf Growth
Parameters
LAR (m2 g21)
SLA (m2 g21)
LWR (g g21)
a
ANOVAa
Treatment
LCHN
HCHN
LCLN
HCLN
CO2
N
0.017 6 0.0005
0.034 6 0.0008
0.51 6 0.007
0.013 6 0.0003
0.026 6 0.0006
0.48 6 0.005
0.016 6 0.0008
0.030 6 0.0006
0.52 6 0.025
0.011 6 0.0002
0.021 6 0.0004
0.51 6 0.004
***
***
**
***
ANOVA, Analysis of variance: **, P , 0.01; ***, P , 0.001. There were no significant interactions between CO2 and N.
suggesting increased control by ribulose-1,5-bisphosphate
regeneration.
In vivo measurements of Rubisco activity modeled from
the A/ci curves show that for the sixth leaf at ligule emergence there was no significant difference in Vc,max between
the CO2 or N treatments (Fig. 2; F 5 0.170, 0.170; P . 0.65).
This was confirmed by in vitro measurements, which also
found no significant difference in the initial activity (Fig. 4;
F 5 0.032, 2.218; P . 0.1) or the activated activity of
Rubisco (Fig. 4; F 5 0.510, 1.449; P . 0.25). There was also
no change in the quantity of this enzyme (Fig. 4; F 5 1.205,
2.651; P . 0.1).
When in vivo Rubisco activity was estimated for wheat
grown in the sand-culture experiment, it was found that for
the fourth and sixth leaves at ligule emergence there was a
significant effect of both pCO2 and N on Vc,max (Fig. 2; F 5
13.326, 13.469; P ,0.01). The Vc,max of the elevated pCO2,
low-N-grown plants was significantly less than for those
grown at control pCO2 and low N (Fig. 2; P ,0.01), indicating a significant decrease in the amount of active
Rubisco.
The operating point on the A/ci curve of the hydroponically grown plants at elevated pCO2 was at the inflection
of the curve and so would be partially influenced by the
rate of regeneration of ribulose-1,5-bisphosphate and ultimately on the rate of electron transport. The rates of Amax
for the sixth leaves show that although a depression was
indicated for leaves grown at elevated pCO2 with low N,
there was no significant effect of pCO2 or N treatment (Fig.
5; F 5 0.379, 4.592; P . 0.05). To verify that triose-3 phosphate export from the chloroplasts was not limiting to leaf
gas exchange the pO2 response of photosynthesis was investigated. Plants responded positively to a reduction in
the pO2 from 21 to 2.1 kPa and showed no pCO2 or N
treatment effect (Fig. 5; F 5 3.333, 0.146; P . 0.05). The
ability of all plants to respond similarly to the removal of
photorespiration consequently supports the Amax results.
DISCUSSION
Acclimation of photosynthesis to elevated pCO2 was
accentuated by low-N supply when wheat was grown in
pots with a fixed [N]. However, when N was supplied in
direct proportion to plant growth, elevated pCO2 did not
produce acclimation of photosynthesis regardless of
whether the N supply was strongly limiting growth or
optimal. Pettersson et al. (1993), who have previously used
the RAR for applying N, have obtained similar findings
with birch. The data support the hypothesis that acclimation of photosynthesis to elevated pCO2 results from a
greater dilution of plant N content rather than from a low
availability of N. Our results suggest that if a plant commences development with a low availability of N, paralleling a plant germinating on a N-deficient soil, the major
effect will be on the rate of leaf-area development rather
than on leaf N content (Scott et al., 1994). As the root
system expands, further N may become available, a situation that may be simulated by the RAR method. When a
plant germinates within a pot with N supplied at a fixed
concentration, as simulated by our sand-culture experiment, initially there is a high availability of N relative to
plant mass, allowing rapid growth. However, with further
growth the relative amount of N will decline and trigger
acclimation of photosynthetic capacity. A corresponding
situation may occur in the field, when over-winter mineralization or fertilizer application at sowing creates a flush
of available N followed by a depletion of the N reserve
through the season.
The dilution effect on plant N status produced by growth
in elevated pCO2 can result either from increasing dilution
of a given N supply (Coleman et al., 1993) or by increased
carbohydrate accumulation diluting the [N] within the
plant (Wong, 1990; Kuehny et al., 1991). Complications can
arise if changes in SLA are not taken into account. This is
because elevated pCO2 frequently alters leaf morphology
Table III. Leaf tissue N content of sand-grown wheat
Leaf tissue N content of sand-culture-grown wheat at 360 mmol mol21 CO2 (LC) or 650 mmol mol21 CO2 (HC) with either 10 mmol of nitrate
(HN) or 4.5 mmol of nitrate (LN). Analyses are for the third leaf at ligule emergence. Values shown are the mean 6 SE per unit dry weight;
n 5 10.
a
ANOVAa
Treatment
Tissue N
Concentration
LCHN
HCHN
LCLN
HCLN
Leaf (mg g21)
Leaf (g m22)
57.1 6 2.5
1.7 6 0.1
54.4 6 1.5
1.9 6 0.1
28.8 6 1.2
1.1 6 0.1
17.2 6 0.8
0.7 6 0.0
ANOVA, Analysis of variance: ***, P , 0.001. There were no significant interactions between CO2 and N.
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CO2
N
***
***
***
578
Farage et al.
Figure 3. CO2-response curves for the sixth leaf at ligule emergence
of wheat grown hydroponically with free access to N or with a RAR
of 0.07 mol N mol21 N d21 in atmospheres of 360 or 650 mmol
mol21 CO2. Symbols are data points from two representative leaves
used in the calculation of Vc,max and Amax, whereas the curves are
fitted by a maximum likelihood regression using the equations of
Farquhar et al. (1980) to all of the leaves measured. The supply
functions are indicated by dotted lines. a, 360 mmol mol21 CO2, free
access to N; b, 650 mmol mol21 CO2, free access to N; c, 360 mmol
mol21 CO2, RAR of 0.07 mol N mol21 N d21; and d, 650 mmol
mol21 CO2; RAR of 0.07 mol N mol21 N d21. Measurement conditions are described in Figure 2.
and, consequently, effects of pCO2 on [N] are decreased
(Norby et al., 1992; Rogers et al., 1996a) or absent
(Rowland-Bamford et al., 1991) when results are expressed
per leaf area rather than leaf weight. Our hydroponically
grown wheat did show an increase in the C-to-N ratio, but
the effect was relatively small and leaf N content remained
above 1.8 g m22. It is well established that rates of photosynthesis are correlated to N content, but for wheat, this
relationship begins to plateau at leaf concentrations above
1.75 g m22 (Evans, 1989). In another investigation, when
growth techniques were compared the C-to-N ratio of tobacco plants was markedly increased at elevated pCO2
when they were grown in pots but was largely unchanged
following growth in hydroponic culture (Ferrario-Méry et
al., 1997).
Availability of N is also a crucial factor in sink development. Rogers et al. (1996b) have shown that the degree of N
fertilization is an important contributor to sink strength,
demonstrating that it can prevent acclimation at all but the
lowest rates of N application. In relation to wheat, Rogers
et al. (1996a) have demonstrated the requirement of N for
tiller and leaf production for avoidance of acclimation.
Similar conclusions were obtained by Ryle et al. (1992) and
by Newbery and Wolfenden (1996). However, in our hydroponics experiment both leaf area and the number of
tillers were significantly reduced by the low-N RAR at both
Plant Physiol. Vol. 118, 1998
control and elevated pCO2, but acclimation was still
avoided. This demonstrates that production of a large sink
capacity may not be necessary to avoid acclimation, rather,
the balance between source and sink at the whole-plant
level is the key factor, as has been proposed by Pettersson
and McDonald (1994). Thus, at elevated pCO2, although
our hydroponically grown, low-N wheat plants maintained
their photosynthetic rates, the absolute amount of photosynthate produced was lessened because of their smaller
leaf area (data not shown) compared with that of their
high-N-grown counterparts. In addition, the ability of the
hydroponically grown wheat to respond to a lowering of
the pO2 demonstrates that the requirement for Pi by ATP
phosphorylase was not greater than the rate of sugar phosphate use (Sharkey, 1985). An interesting observation,
however, was that the hydroponically grown wheat had a
greater mass than those plants grown in ambient air, despite there being no significant effect of pCO2 on relative
growth rate. This difference must have been initiated from
a very early and transient stimulation of growth, and consequently appears similar to observations made in other
elevated-CO2 studies where differences in biomass between pCO2 treatments are reported to have arisen from
brief alterations in relative growth rate (Poorter, 1993).
Acclimation of photosynthesis to growth in elevated
pCO2 is most frequently accompanied by a reduction in
carboxylation capacity (for review, see Bowes, 1991). It is
commonly suggested that a decrease in Rubisco activity is
ultimately responsible for acclimation of photosynthesis to
elevated pCO2 (Bowes, 1991). Estimations of Vc,max in vivo
and direct measurement of Rubisco activity and content in
vitro confirmed that the pCO2 and N treatments had no
significant effect on the hydroponically grown wheat, and
this is the most likely reason that net photosynthesis was
Figure 4. Vc,max of Rubisco measured in vitro together with the
concentration of Rubisco protein. Activities are for the initial activity
upon extraction and for the Vc,max following incubation with CO2
and Mg21. Legend for bars is in Figure 2; vertical bars represent SE;
n 5 4.
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Photosynthetic Acclimation and Nitrogen Supply
Figure 5. The rate of CO2 uptake at light and CO2 saturation (Amax)
and the relative stimulation (stim.) of CO2 uptake by inhibition of
photorespiration following a reduction in pO2 to 21 mmol mol21.
Measurement conditions for leaf gas exchange and legends for bars
are described in Figure 2; the [CO2] was 650 mmol mol21. Vertical
bars represent SE; n 5 4.
579
do not keep pace with demand and, therefore, the plants
will be prone to acclimation. The experimental approach
that has least often found acclimation is the field system
(Gunderson and Wullschleger, 1994; Sage, 1994; Drake et
al., 1997). Plants growing in the natural environment are
more likely to be adjusted to surrounding environmental
influences. However, this does not preclude acclimation
from occurring; natural environments are often limiting in
nutrients, especially N (Eamus and Jarvis, 1989; Gifford,
1992), and, consequently, there is ample opportunity for
acclimation of photosynthesis to occur. Plants can experience periods of source:sink imbalance, especially late in the
growing season when reproductive sinks are developing.
For example, translocation of N during grain filling was
assumed to produce a decrease in Rubisco concentration of
wheat exposed to elevated CO2 in a free air CO2 enrichment system (Nie et al., 1995), and changes in developmental state have been shown to initiate reversible acclimation
in beet (Ziska et al., 1995). We might therefore expect
phenology, together with specific environmental conditions, to be instrumental in determining the response of
plants to elevated pCO2 in the natural environment.
ACKNOWLEDGMENTS
unaffected. The lack of effect on Rubisco most likely stems
from the absence of either any major reduction in leaf N
content or of any end-product inhibition of photosynthesis.
Increase of leaf carbohydrate has been correlated with a
decrease in Vc,max (McKee and Woodward, 1994), and it is
this increase in photosynthate that is suggested to be the
signal that brings about a decrease in Rubisco levels (Stitt,
1991). The underlying mechanism is believed to operate via
the repression of photosynthetic gene expression (Webber
et al., 1994; Koch, 1996; Drake et al., 1997). Whether the
quantity of Rubisco is decreased at elevated pCO2 in response to buildup in leaf carbohydrate or not, less active
enzyme is required because it is not saturated at the current
atmospheric pCO2 (Long, 1991). Consequently, N may be
reallocated to proteins of the electron-transport chain or
photosynthetic carbon-reduction-cycle enzymes (Woodrow, 1994). Wheat grown under true field conditions (Free
Air CO2 Enrichment) showed no loss of Rubisco activity or
quantity in unshaded leaves, and this was accompanied by
a complete absence of any down-regulation of photosynthesis (Nie et al., 1995; Drake et al., 1997).
In conclusion, the results from our investigation have
shown that low rates of N supply need not cause acclimation of photosynthesis to elevated pCO2. Development of
large sinks, e.g. tillers and leaves, was not necessary for the
avoidance of acclimation; rather, an adjustment of plant
growth rate to match the N supply appears to be the
decisive factor. This agrees with the hypothesis by Pettersson and McDonald (1994) that acclimation to elevated
pCO2 is dependent upon whether the whole-plant growth
response has acclimated to elevated pCO2, together with
any other resource limitations in the immediate environment. The majority of experimental methodologies used in
elevated pCO2 investigations by necessity grow plants in
artificial conditions, but the outcome may be that resources
We thank S. Corbet for technical assistance with the hydroponics culture and P. Beckwith for general technical skills. J. Bullimore’s help with the Rubisco analyses is much appreciated.
Received March 2, 1998; accepted July 7, 1998.
Copyright Clearance Center: 0032–0889/98/118/0573/08.
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