Annals of Botany 88: 571±577, 2001
doi:10.1006/anbo.2001.1503, available online at http://www.idealibrary.com on
In¯uence of High CO2 Partial Pressure on Nitrogen Use Eciency of the C4 Grasses
Panicum coloratum and Cenchrus ciliaris
S ILV IA G . R U D M A N N {, PA UL J. M IL H A M { and JA N N P. CO N ROY * {
{Centre for Horticulture and Plant Sciences, University of Western Sydney, Hawkesbury Campus, Locked Bag 1797,
South Penrith Distribution Centre, NSW 1797, Australia and {NSW Agriculture, Agriculture Institute, Forest Rd.
Orange, NSW 2800, Australia
Received: 16 March 2001 Returned for revision: 15 May 2001 Accepted: 15 June 2001
Australia's tropical grasslands are dominated by C4 grasses, characterized by their unique biochemistry and
anatomy. Two naturalized C4 grasses (Panicum coloratum and Cenchrus ciliaris) were used to investigate whether
high CO2 partial pressure [p(CO2)] in¯uences photosynthetic nitrogen use eciency and plant nitrogen use
eciency (PNUE and NUE respectively). Plants were grown for 30 d with four levels of N at p(CO2) of 38 or
86 Pa. PNUE was calculated from leaf CO2 assimilation rates (A) and leaf N concentrations, and NUE from total
leaf N content and plant dry mass. At each p(CO2), PNUE and NUE were greater for C. ciliaris than for
P. coloratum due to higher A and dry mass combined with lower leaf N concentrations. Elevated p(CO2) increased
PNUE of C. ciliaris only. This e€ect was due to lower leaf N concentrations (area basis). At high p(CO2), NUE of
C. ciliaris was also greater. This resulted from a 1.6-fold stimulation of dry mass by high p(CO2). Although dry
mass of P. coloratum was increased 1.2-fold by elevated p(CO2), its NUE was una€ected. Leaf transpiration rates
were halved at elevated p(CO2), and we suggest that this factor plays a major role in the growth response of C4
# 2001 Annals of Botany Company
grasses to high p(CO2).
Key words: Panicum coloratum, Cenchrus ciliaris, nitrogen use eciency, elevated CO2, leaf N concentration, growth,
photosynthesis.
I N T RO D U C T I O N
Grasses with the C4 photosynthetic pathway dominate
tropical grasslands and account for approximately 20 % of
the present global productivity. The land area occupied by
C4 grasslands is likely to increase in Australia and elsewhere
under the global warming scenario for the 21st century
(Archer, 1993; Henderson et al., 1994). What is less certain is
whether rising atmospheric CO2 partial pressure [ p(CO2)]
will also in¯uence productivity of the C4 grasslands. The
reason for this uncertainty is that relatively few CO2
enrichment studies have focused on the response of C4
grasses to high p(CO2) compared to the large number of
studies on C3 plants (Ghannoum et al., 2000). C4 grasses
have a specialized leaf anatomy and biochemistry that
results in a three- to ten-fold higher concentration of CO2 in
the bundle sheath cells compared to that in C3 plants
(Furbank et al., 1989). Consequently, C4 grasses were not
expected to respond to rising atmospheric p(CO2). In
contrast, for C3 plants, doubling the p(CO2) increases CO2
assimilation rates (A) and this generally explains the 20 to
40 % increase in plant dry mass in response to high p(CO2)
(Kimball, 1983; Poorter, 1993).
Aside from growth stimulation, a key di€erence between
C3 plants grown at di€erent p(CO2) is that elevated p(CO2)
reduces leaf N concentrations (Stitt and Krapp, 1999). The
* For correspondence. Fax 006 02 45701314, e-mail jp.conroy@uws.
edu.au
0305-7364/01/100571+07 $35.00/00
magnitude of the decrease is related to N status. In wheat, for
example, N concentration (on a dry mass basis) decreased
by 30 and 7 % at low and luxury N supplies, respectively
(Rogers et al., 1996). Consequently, nitrogen use eciency
(NUE) and photosynthetic nitrogen use eciency (PNUE)
are greater at elevated p(CO2) (Evans, 1989; Oaks, 1994;
Aben et al., 1999), i.e. lower leaf N concentrations are
required to support maximum growth and A (Rogers et al.,
1996; Aben et al., 1999). Should similar responses occur in
C4 plants, the productivity of tropical grasslands may
increase as p(CO2) rises because their growth is currently
N-limited (Howden et al., 1999). Importantly, the leaf N
concentration below which growth slows is a key factor in
models of productivity of tropical grasslands (Howden et al.,
1999).
At the current p(CO2) and a given N supply, C4 plants
have a higher NUE than C3 plants. The higher NUE of the
former results from a lower investment of leaf N in Rubisco
protein compared to C3 plants (Brown, 1978; Pheloung and
Brady, 1979). For C3 grasses, future rises in p(CO2) are likely
to reduce the N concentration (Stitt and Krapp, 1999). The
question is whether N concentrations in leaves of C4 grasses
will also be reduced. There are con¯icting results in the
literature regarding the in¯uence of high p(CO2) on N
concentration in leaves of C4 plants. Reduced foliar N
concentration is reported in some studies (Morgan et al.,
1994; Owensby et al., 1994; Read and Morgan, 1996), but
not in others (Ghannoum et al., 1997; Ghannoum and
Conroy, 1998).
# 2001 Annals of Botany Company
572
Rudmann et al.ÐHigh p(CO2) and Nitrogen Use Eciency of C4 Grasses
If N concentrations are una€ected by rising p(CO2),
NUE of C4 plants could still be increased if plant dry mass
increases. Contrary to earlier hypotheses that growth of C4
grasses would not change at high p(CO2), reviews by
Poorter (1993), Wand et al. (1999) and Ghannoum et al.
(2000) indicate that the growth response of C4 plants to a
doubling of the current p(CO2) ranges from 22 to 33 %. The
most frequent explanations given for increased growth of C4
plants at elevated p(CO2) are that A is stimulated (Wong,
1979; Morgan et al., 1994; Ziska and Bunce, 1997; Le Cain
and Morgan, 1998; Wand et al., 1999; Ziska et al., 1999)
and/or that transpiration rates (E) are reduced (Ghannoum
et al., 2001). A reduction in E results in improved shoot
water relations (Samarakoon and Gi€ord, 1996; Seneweera
et al., 1998) and increased leaf temperature (Ghannoum et
al., 2000).
Not only are there biochemical and anatomical di€erences between C3 and C4 plants, but diversity within C4
species may also in¯uence growth, photosynthesis and
NUE at di€erent p(CO2). Based on their anatomy and
biochemistry, C4 grasses are grouped into three subtypes
named after the major C4 acid decarboxylation enzyme in
the bundle sheath cell: NAD-malic enzyme (NAD-ME);
NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK). In Australia, NAD-ME
and NADP-ME are the dominant subtypes, and NADPME grasses are the more prevalent in areas of above
average annual rainfall (Hattersley, 1992). Bowman (1991)
showed that NUE and PNUE were generally greater for
NADP-ME than for NAD-ME species. The few CO2
enrichment studies that compare plants belonging to the
di€erent C4 subtypes have yielded di€erent results. Ghannoum and Conroy (1998) demonstrated that dry mass of
both NAD-ME and NADP-ME Panicum grasses increased
1.28-fold with elevated CO2, with little change in A. In
contrast, Le Cain and Morgan (1998) found a higher dry
mass and A in NADP-ME species only.
In this paper we investigate whether PNUE and NUE of
C4 grasses are increased by growth at elevated p(CO2).
Panicum coloratum (NAD-ME) and Cenchrus ciliaris
(NADP-ME) were grown for 30 d under controlled
conditions with four levels of N at a p(CO2) of either 38
or 86 Pa. These two species were chosen because they are
relatively fast growing and belong to the same subfamily
and tribe (Panicoidae; Paniceae). Both are naturalized in
Australia and have a perennial growth habit.
(70) and CuSO4.5H2O (20). Approximately ten seeds per pot
of P. coloratum `makarikiense' (NAD-ME subtype) were
sown in each of 56 pots. There were also 56 pots of C. ciliaris
(NADP-ME subtype). Half the pots for each species were
transferred to a growth chamber (Thermoline, Sydney,
Australia) maintained at 38 Pa CO2. The other half were
placed in a similar chamber maintained at 86 Pa CO2 by
injecting CO2 gas from a pressurized cylinder (Food grade,
CIG, Sydney, Australia) through a solenoid valve connected
to an infrared gas analyser (16/32 ICAM, Melborne,
Australia). CO2 was passed through a Pura®l column to
remove any ethylene contamination. During the 12 h light
period, a PPFD of 1000 mmol m ÿ2 s ÿ1 was supplied by
metal halide lamps (GEMVR 1000 U, General Electric,
Sydney, Australia) and incandescent bulbs. The temperature
in the cabinet was 30/25 8C day/night, respectively. The
VPD was 2.1 Pa during the light period.
Seven days after sowing (DAS), the number of plants was
reduced to one per pot for C. ciliaris and two per pot for
P. coloratum. Di€erent numbers of plants were grown for
each species to minimize di€erences between species in leaf
area per pot. Pebbles were placed on top of the soil to
reduce evaporation. N treatments then commenced with
addition rates (mg N kg ÿ1 soil per week) of 0, 20, 40 and 60
for both species (based on a preliminary study). There were
four pots (replicates) of each N treatment at each p(CO2).
Water was supplied via drippers at a rate of 3 l d ÿ1 per pot
split into two 1.5 l applications at the beginning and in the
middle of the light period.
Gas exchange measurements
At 23 DAS A and E were measured using a portable
photosynthesis system (LI-6400, LI-COR, Lincoln, USA).
Measurements were carried out on a section (3 cm2) of the
last fully expanded leaf attached to the main stem (one plant
per pot) for each of the four replicates per species. The
temperature of the leaf, measured by a thermocouple,
ranged from 29.5 to 30.5 8C. PPFD, supplied by an inbuilt LED lamp, was 1000 mmol photons m ÿ2 s ÿ1.
Measurements were made at the p(CO2) at which the plants
were grown. The leaf used for gas exchange measurements
was sampled for analysis of N concentration. Photosynthetic water use eciency (PWUE) was calculated by
dividing A by E.
M AT E R I A L S A N D M E T H O D S
Plant growth
Growth measurements
A sandy loam soil was mixed with the following basal
nutrients (g kg ÿ1 soil): CaCO3 (4), CaHPO4.2H2O (1.5) and
CaSO4 (1.2) prior to transferring 6.5 kg of the mixture to
10 l bottom-drained pots. Rapid mineralization of organicN to NO3 ÿ -N occurred on wetting the soil, and each pot was
therefore leached with water until the NO3 ÿ -N concentration in the leachate was below 5 mg l ÿ1. Micronutrients
were then added in solution (mg metal kg ÿ1 soil) as the
following salts: MgSO4 (57); ZnSO4.7H2O (100); K2CO3
Plants were harvested at 30 DAS. Shoots were cut at the
base of the stems and their lengths measured. Individual
shoots were separated into leaves and stems (stems plus
sheaths). Total leaf area (TLA) was measured using a digital
image analyser (Delta-T, Cambridge, UK). Roots in each
pot were washed free of soil. All plant parts were dried at
80 8C for 48 h prior to determination of their dry mass
(DM). Speci®c leaf area (SLA) was calculated as TLA
(dm2)/DM (g).
Rudmann et al.ÐHigh p(CO2) and Nitrogen Use Eciency of C4 Grasses
Photosynthetic NUE and NUE (leaf basis)
PNUE was calculated from A and leaf N concentration
(area basis) and expressed as mmol CO2 g ÿ1 N s ÿ1. NUE
was calculated from plant mass and total leaf N content
and expressed as g DM g ÿ1 N.
Statistical analysis
Data were analysed using SAS statistics software (SAS
Institute, Cary, North Carolina, USA, 1994). A completely
randomized experimental design was assumed with four
replicates for each N treatment. The signi®cance of
di€erences between the treatments was analysed by
ANOVA and Duncan's multiple range tests. Standard errors
of the means and LSD (P ˆ 0.05) are shown where
appropriate.
R E S U LT S
Growth and morphology
Plant dry mass di€ered between species and as a result of
the CO2 and N treatments (P 5 0.001) (Fig. 1). C. ciliaris
had a consistently higher dry mass than P. coloratum and,
for both species, elevated p(CO2) and increasing N supply
enhanced dry mass (Fig. 1). Overall, the CO2 e€ect was
more pronounced for C. ciliaris (average response 1.6-fold,
maximum 2.6) than for P. coloratum (average response 1.2fold, maximum 1.5). Importantly, high p(CO2) only
increased the dry mass of P. coloratum at 20 mg N kg ÿ1
soil per week, whereas growth of C. ciliaris was increased by
high p(CO2) at all N treatments.
Enhanced dry mass production at higher N supplies
(averaged across CO2 treatments) was also greater for
C. ciliaris (3-fold increase) than for P. coloratum (2-fold
increase). The shape of the growth responses to N for both
C4 grasses was similar at both CO2 treatments, i.e. there was
an increase in mass until 20 mg N kg ÿ1 soil per week and
no signi®cant response thereafter (Fig. 1). The exception
was P. coloratum grown at 38 Pa CO2, where the increments
in mass continued until 60 mg N kg ÿ1 soil per week
(Fig. 1). Dry mass partitioning varied between species and,
on average, C. ciliaris allocated more dry mass to the stem
plus sheath (42 %) than did P. coloratum (27 %).
There were morphological responses of both species to
high p(CO2) and increasing N supply. Higher N and
elevated p(CO2) enhanced the shoot length by up to 1.2-fold
for both C4 grasses (P 5 0.01) (Table 1). For both species,
Gas exchange
A was greater for C. ciliaris than for P. coloratum in all N
and CO2 treatments (Table 2); however, the photosynthetic
responses di€ered markedly from growth responses to these
treatments (Fig. 1, Table 2). Importantly, there was no
signi®cant e€ect of high p(CO2) on A for either species, and
the response of A to higher N supplies was smaller than the
growth response (Fig. 1, Table 2). The greatest stimulation
of A was between 0 and 20 mg N kg ÿ1 soil per week (1.5and 1.3-fold increase for P. coloratum and C. ciliaris,
respectively). In contrast to its lack of e€ect on A, elevated
p(CO2) approximately halved E in both species (Fig. 2).
The magnitude of E was 1.6-times greater for C. ciliaris
than for P. coloratum (Fig. 2).
Mineral nutrition
The C4 species di€ered in their leaf N concentrations and
in their response to CO2 and N treatments. An important
30
Total dry mass (g per plant)
Dried samples of the plant parts were ground in a
Cyclotec mill (Tecator, HoganaÈs, Sweden). Mineral nutrient concentrations in leaves (P, K, Ca, Mg, S, Fe, Mn, B,
Cu, and Zn) were measured by ICP-MS (Thermo-Optek,
Winsford, UK) after digesting the ground samples in
HNO3. N concentration of each plant fraction was analysed
using an N analyser (Leco FP-428, St. Joseph, USA). The
N concentration of the last fully expanded leaf sampled at
23 DAS (leaf used for gas exchange) was also measured.
increases in total leaf area followed the same pattern as that
of dry mass (Fig. 1, Table 1). Consequently, speci®c leaf
area was una€ected by high p(CO2) (P 4 0.05) and was
enhanced by increasing N additions (Table 1).
25
20
15
10
5
Leaf N concentration (mg N kg-1 LDM)
Mineral nutrients
573
0
50
40
30
20
10
0
0
20
40
60
N supply (mg N kg-1 soil per week)
F I G . 1. Plant dry mass and leaf N concentration (dry mass basis) of
C. ciliaris (q, Q) and P. coloratum (w, W) grown at either 38 (q, w) or
86 (Q, W) Pa CO2 for 30 DAS. Values are means of four replicates.
Bars represent two standard errors.
574
Rudmann et al.ÐHigh p(CO2) and Nitrogen Use Eciency of C4 Grasses
T A B L E 1. Growth parameters of P. coloratum (NAD-ME) and C. ciliaris (NADP-ME)
N supply (mg N kg ÿ1 soil per week)
CO2 (Pa)
0
20
40
60
LSD (P ˆ 0.05)
TLA*
(dm2)
SLA
(dm2 g ÿ1)
Shoot length
(cm)
38
86
38
86
38
86
2 .3
3 .6
1 .5
1 .8
57
71
8.8
11.1
3.2
2.6
69
89
11.8
11.4
3.1
3.2
82
85
14.2
14.9
4.3
4.8
70
80
0.9
TLA
(dm2)
SLA
(dm2 g ÿ1)
Shoot length
(cm)
38
86
38
86
38
86
3 .7
5 .8
2 .1
2 .5
30
50
15.3
21.9
3.2
3.2
49
62
19.2
32.3
2.8
3.5
53
54
21.5
36.0
3.6
3.9
60
74
Species
Parameters
P. coloratum
C. ciliaris
0.4
6.0
1.3
0.4
1.5
Plants were grown at 38 and 86 Pa CO2 and with di€erent N supplies for 30 d. Values are the means of four replicates.
*TLA, Total leaf area; SLA, speci®c leaf area.
T A B L E 2. Leaf N concentration, A and PWUE of P. coloratum (NAD-ME) and C. ciliaris (NADP-ME)
N supply (mg N kgÿ1 soil per week)
Species
P. coloratum
C. ciliaris
CO2 (Pa)
0
20
40
60
LSD (P ˆ 0.05)
Leaf N concentration
(mmol N m ÿ2)
38
86
72.6
89.6
74.7
81.6
86.7
84.9
78.6
80.6
12.6
A*
(mmol CO2 m ÿ2 s ÿ1)
38
86
25
26
34
32
37
40
35
33
3.6
PWUE
(mmol CO2 mmol ÿ1 H2O)
38
86
8
20
8
19
7
17
8
18
1.2
Leaf N concentration
(mmol N m ÿ2)
38
86
48.5
38.5
52.4
49.5
84.4
60.6
89.5
60.7
A
(mmol CO2 m ÿ2 s ÿ1)
38
86
36
37
45
45
43
52
42
42
4.5
PWUE
(mmol CO2 mmol ÿ1 H2O)
38
86
8
18
7
14
8
13
7
14
1.6
Parameters
12.5
Plants were grown at 38 and 86 Pa CO2 and with di€erent N supplies for 30 d. Values are the means of four replicates.
*A, CO2 assimilation rate; PWUE, photosynthetic water use eciency.
observation was that leaf N concentrations (expressed on
either a dry mass or area basis) were consistently higher for
P. coloratum than for C. ciliaris in all treatments (Fig. 1,
Table 2). This occurred for the bulk leaf fraction at 30 DAS
and the last fully expanded leaves at 23 DAS, there being no
signi®cant di€erence in N concentrations between these leaf
fractions. Higher N supplies increased the N concentration
in leaves (dry mass basis) of both species (P 5 0.001).
Elevated p(CO2) had no signi®cant e€ect on leaf N
concentration (P 4 0.05) estimated on a dry mass basis
(Fig. 1). However, on a leaf area basis, N concentrations of
leaves of C. ciliaris tended to be lower at elevated p(CO2)
(Table 2). Concentrations of P, K, Ca, Mg, S, Fe, Mn, B,
Cu, and Zn were una€ected by the treatments (data not
shown). Comparison of the concentrations of these
nutrients with adequate concentrations for Zea mays
(NADP-ME) and C. ciliaris (NADP-ME) indicated that
the nutrition was satisfactory to support maximum growth
(Reuter et al. 1997).
Nitrogen and water use eciency
Plant and photosynthetic NUE was generally greater for
C. ciliaris than for P. coloratum, irrespective of the basis on
which it was calculated (PNUE or NUE) (Fig. 3). There
was a tendency for PNUE to increase with high p(CO2) in
C. ciliaris only; however, the CO2 e€ect was not signi®cant
for either species (Fig. 3). PNUE di€ered between the
species with signi®cantly higher (50 %) values for C. ciliaris
than for P. coloratum. Mean values of PNUE were 315 and
560 mmol CO2 g ÿ1 N s ÿ1 for P. coloratum and C. ciliaris,
respectively. NUE was greater at high p(CO2) in C. ciliaris
only. Importantly, both species had similar NUE at the
highest N supply, irrespective of CO2 treatments (Fig. 3).
Rudmann et al.ÐHigh p(CO2) and Nitrogen Use Eciency of C4 Grasses
PNUE ( mol CO2 mol
_
1
leaf N s
_
1
)
30
1000
800
600
400
2
m
_
2
s
_
1
)
20
E (mmol H O m
575
5
200
0
N)
350
0
0
20
N supply (mg N kg
40
_
1
60
soil per week)
F I G . 2. Transpiration rates (E) of last fully expanded leaves of
C. ciliaris (h, j) and P. coloratum (s, d) at 23 DAS. Growth and
gas exchange were measured at either 38 (w, h) or 86 Pa CO2 (d, j).
Values are the means of four replicates. Bars represent two standard
errors.
PWUE doubled at high p(CO2) in both species (Table 2).
At 38 Pa CO2, no di€erences in PWUE between the N
treatments were observed; however, at 86 Pa CO2, higher N
supplies tend to reduce PWUE in both C4 grasses (Table 2).
DISCUSSION
The C4 grasses, P. coloratum and C. ciliaris, di€ered in their
growth and photosynthetic responses to elevated p(CO2).
These di€erences were re¯ected in the response of PNUE
and NUE of each species to high p(CO2). For P. coloratum,
high p(CO2) had no signi®cant e€ect on PNUE because
neither A nor leaf N concentrations were in¯uenced by
p(CO2) (Fig. 3, Table 2). However, PNUE of C. ciliaris
tended to be higher at elevated p(CO2) because of a small
reduction in leaf N concentration (area basis), especially at
high N supplies (Fig. 3, Table 2). Elevated p(CO2)
increased plant NUE of C. ciliaris because of the large
(1.6-fold on average) stimulation of dry mass by high
p(CO2) (Figs 1 and 3). Signi®cant CO2 e€ects were observed
at all N treatments for this species. In contrast, there was no
stimulation of plant NUE by elevated p(CO2) in
P. coloratum, and the e€ect of CO2 was signi®cant at the
20 mg N kg ÿ1 soil per week treatment only (Fig. 1).
Our results di€er from those obtained for C3 plants, in
which PNUE and NUE have been shown to increase at
elevated p(CO2) (Evans, 1989; Oaks, 1994). Our ®ndings
emphasize the variation between C3 and C4 plants, and
NUE (g plant dry mass g
_
1
2
300
250
200
150
100
50
0
0
20
N supply (mg N kg
40
_
1
60
soil per week)
F I G . 3. NUE and PNUE of C. ciliaris (q, Q) and P. coloratum (w, W)
grown at either 38 (q, w) or 86 (Q, W) Pa CO2 for 30 DAS. Values are
means of four replicates. Bars represent two standard errors.
within C4 species, in the physiological basis of the CO2
response. The dry mass of both our C4 subtypes was
increased by high p(CO2) in the absence of a photosynthetic
response (Fig. 1, Table 2). This is a major di€erence to C3
plants, in which A is generally enhanced by elevated p(CO2)
although, in the long-term, there may be photosynthetic
acclimation to high p(CO2) (Stitt and Krapp, 1999). A
further contrast to C3 plants was that high p(CO2) had little
e€ect on leaf N concentrations in our C4 grasses (Fig. 1,
Table 2). This is in agreement with some (Ghannoum and
Conroy, 1998), but not all (Le Cain and Morgan, 1998)
studies of C4 plants. Accumulation of soluble carbohydrates
accounted for the lower N concentrations in leaves of
NADP-ME grasses at high p(CO2) in the experiment of Le
Cain and Morgan (1998). In contrast, elevated p(CO2) had
no e€ect on soluble carbohydrate concentrations in leaves of
either NAD-ME or NADP-ME subtypes (Ghannoum and
Conroy, 1998; Rudmann, 2000). The reduction in leaf N
concentration in C3 plants is attributed to lower Rubisco
concentrations in leaves (Rogers et al., 1996; Nakano et al.,
1997). There have also been suggestions that reductions in E
at high p(CO2) may cause a lower ¯ux of N through the soil
to the root surface, thereby reducing N uptake (Conroy,
1992). The data in this experiment do not support this
576
Rudmann et al.ÐHigh p(CO2) and Nitrogen Use Eciency of C4 Grasses
suggestion because E was halved by elevated p(CO2) with no
e€ect on leaf N concentration (Figs 1 and 2).
There was a striking di€erence between species belonging
to the di€erent C4 subtypes, with C. ciliaris (NADP-ME)
having a markedly higher PNUE and NUE than
P. coloratum (NAD-ME) (Fig. 3, Table 2). This resulted
from lower leaf N concentrations and higher A and dry mass
for C. ciliaris than for P. coloratum (Fig. 1, Table 2).
Bowman (1991) also showed that NUE and PNUE were
greater for two NADP-ME than four NAD-ME Panicum
species. Other studies have shown that leaf N concentrations
are generally lower for NADP-ME subtypes (Pheloung and
Brady, 1979; Le Cain and Morgan, 1998). The physiological
basis for this di€erence is unknown; however, variations in
anatomy, organelle distribution and biochemistry are likely
to contribute. As a result of the di€erence in N concentrations between species, growth of C. ciliaris slowed at a
lower N concentration (1.6 mol N g ÿ1 dry mass) than for
P. coloratum (2.3 and 2.7 mol N g ÿ1 dry mass at 38 and
86 Pa CO2). This could have implications for competition of
C4 grasses in tropical pastures.
The results of our study raise questions about the
mechanism underlying the growth response of C4 species
to high p(CO2). We conclude that large reductions in
transpiration rate at high p(CO2) played a major role in
stimulating growth of P. coloratum and C. ciliaris at elevated
p(CO2) (Figs 1 and 2). It is particularly important to note
that C. ciliaris had a higher E than P. coloratum and that
elevated p(CO2) reduced E of C. ciliaris to values close to
those measured for P. coloratum at 38 Pa CO2 (Fig. 2).
Previous experiments with P. coloratum and Zea mays have
demonstrated that both low soil water status and high E
reduced leaf growth and that the e€ects of both stresses were
additive (Ben Haj Salah and Tardieu, 1997; Seneweera et al.,
1998). In P. coloratum, elevated p(CO2) ameliorated these
stresses by maintaining higher leaf water potentials and
reducing transpiration (Seneweera et al., 1998). In our
experiment, shoot length was greater at elevated p(CO2)
(Table 1). This supports the suggestion that leaf water
relations and/or lower E were responsible for the growth
stimulation by elevated p(CO2) because both stem and leaf
growth are sensitive to mild soil water de®cits and high E
(Ben Haj Salah and Tardieu, 1997). We found no increases
in A due to p(CO2), when measurements were made at
30 + 0.5 8C using gas exchange (Table 2). However, this
does not rule out the possibility that during the growth of
the plants, A may have been higher at elevated p(CO2) due to
higher leaf temperatures, induced by a lower E (Fig. 2). This
is possible because the optimum leaf temperature for A for
C4 grasses is above 30 8C (Ghannoum et al., 2001).
AC K N OW L E D G E M E N T S
We thank Dr Oula Ghannoum for helpful discussions and
CSIRO Division of Tropical Crops and Pastures (St. Lucia,
Queensland) for the supply of seeds of the C4 grasses. This
research was supported by Large Australian Research
Council Grant number A19906169.
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