Journal of Experimental Botany, Vol. 62, No. 9, pp. 3223–3234, 2011
doi:10.1093/jxb/err005 Advance Access publication 9 February, 2011
RESEARCH PAPER
Panicum milioides (C3–C4) does not have improved water
or nitrogen economies relative to C3 and C4 congeners
exposed to industrial-age climate change
Harshini Pinto, David T. Tissue and Oula Ghannoum*
Hawkesbury Institute for the Environment, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2753, Australia
* To whom correspondence should be addressed. E-mail: [email protected]
Abstract
The physiological implications of C3–C4 photosynthesis were investigated using closely related Panicum species
exposed to industrial-age climate change. Panicum bisulcatum (C3), P. milioides (C3–C4), and P. coloratum (C4) were
grown in a glasshouse at three CO2 concentrations ([CO2]: 280, 400, and 650 ml l21) and two air temperatures
[ambient (27/19 C day/night) and ambient + 4 C] for 12 weeks. Under current ambient [CO2] and temperature, the
C3–C4 species had higher photosynthetic rates and lower stomatal limitation and electron cost of photosynthesis
relative to the C3 species. These photosynthetic advantages did not improve leaf- or plant-level water (WUE) or
nitrogen (NUE) use efficiencies of the C3–C4 relative to the C3 Panicum species. In contrast, the C4 species had
higher photosynthetic rates and WUE but similar NUE to the C3 species. Increasing [CO2] mainly stimulated
photosynthesis of the C3 and C3–C4 species, while high temperature had no or negative effects on photosynthesis of
the Panicum species. Under ambient temperature, increasing [CO2] enhanced the biomass of the C3 species only.
Under high temperature, increasing [CO2] enhanced the biomass of the C3 and C3–C4 species to the same extent,
indicating increased CO2 limitation in the C3–C4 intermediate at high temperature. Growth [CO2] and temperature
had complex interactive effects, but did not alter the ranking of key physiological parameters amongst the Panicum
species. In conclusion, the ability of C3–C4 intermediate species partially to recycle photorespired CO2 did not
improve WUE or NUE relative to congeneric C3 or C4 species grown under varying [CO2] and temperature conditions.
Key words: C3, C4, and C3–C4 photosynthesis, climate change, Panicum, water and nitrogen use efficiency.
Introduction
C4 photosynthesis evolved from C3 photosynthesis some
20–30 million years ago to overcome the inefficiencies of the
primary CO2-fixing enzyme in plants, Rubisco (ribulose-1,5bisphosphate carboxylase/oxygenase), particularly at low
atmospheric CO2 concentration ([CO2]) and warm temperatures (Sage, 2004). The key novelty of C4 photosynthesis is
the employment of spatially separated carboxylation and
decarboxylation reactions. The initial carboxylation reaction
is catalysed by phosphoenolpyruvate carboxylase (PEPC) and
involves the transient incorporation of inorganic carbon into
organic C4 acids in the mesophyll cell (MC). This is followed
by the decarboxylation of C4 acids in the bundle sheath cell
(BSC) and the release of CO2 for permanent fixation into
organic C3 acids in a reaction catalysed by Rubisco. The fast
delivery of CO2 by the C4 cycle leads to high [CO2] in the
BSC which serves to saturate the carboxylation and suppress
the oxygenation reactions of Rubisco under current ambient
[CO2] (Hatch, 1987). The high BSC [CO2] and the suppression
of photorespiration have well-recognized effects on the
physiology and ecophysiology of C4 plants (Brown, 1978;
Osmond et al., 1982; Ehleringer and Monson, 1993; Long,
1999; Ghannoum et al., 2011).
Importantly, high BSC [CO2] saturates C4 photosynthesis
at relatively low intercellular [CO2] (Ci), allowing C4 plants
to operate with lower stomatal conductance (gs). Thus, leaflevel photosynthetic water-use efficiency (PWUE, rate of
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
Downloaded from http://jxb.oxfordjournals.org/ at OUP site access on April 22, 2013
Received 5 November 2010; Revised 4 January 2011; Accepted 7 January 2011
3224 | Pinto et al.
C3–C4 relative to C3 species, except for small improvements
in PWUE and PNUE under low [CO2] (Ku and Edwards,
1978; Brown and Simmons, 1979; Bolton and Brown, 1980).
Therefore, it remains unclear whether the type of C3–C4
photosynthesis present in intermediate Panicum species leads
to detectable improvements in PWUE and PNUE relative to
closely related C3 Panicum species.
In addition to the evolutionary aspect, there is a need to
understand whether physiological advantages conferred by
C4 photosynthesis, and presumably conferred by C3–C4
photosynthesis, hold under predicted future elevated atmospheric [CO2] and high air temperature scenarios. To our
knowledge, there are no published studies investigating the
combined effects of elevated [CO2] and high temperature or
the effects of subambient [CO2] on the physiology of C3–C4
intermediate species. Consequently, the current study was
undertaken to compare the photosynthetic and whole-plant
WUE and NUE of closely related C3, C4, and C3–C4 Panicum
species under pre-industrial, current ambient, and projected
mid-21st century atmospheric [CO2]. Plants were also exposed
to ambient temperature or ambient temperature + 4 C to
investigate the combined effects of elevated [CO2] and global
warming on the physiology of the Panicum species.
Materials and methods
Plant culture and water use measurements
The experiment was conducted in a naturally lit glasshouse
consisting of six adjacent chambers (;5 m3 each). Three chambers
were maintained at ambient temperature, and three chambers were
maintained at ambient temperature + 4 C. The average day/night
temperatures for ambient and high temperature treatments were
27/19 C and 31/22 C, respectively. The average relative humidity
of the glasshouse rooms was ;75%. The three ambient temperature rooms were maintained at three different [CO2]: subambient
(280 ll l1, pre-industrial), ambient (400 ll l1, current), and
elevated (640 ll l1, predicted for the mid-21st century). The
[CO2] treatments were similar for ambient and high temperature
treatments. A detailed description of the temperature and CO2
control can be found in Ghannoum et al. (2010a).
Polyethylene bags were placed inside 3.5 l cylindrical pots to
prevent water leakage. Pot mass was adjusted to 0.8 kg using
pebbles. Air-dried and coarsely sieved soil (3.7 kg) was added to
each pot. Soil water capacity was calculated as the difference
between the mass of two non-watered pots and that of pots
watered and then left to drain freely overnight. Pots were watered
to 100% capacity then transferred to the six glasshouse chambers.
Seeds for Panicum bisulcatum (C3), Panicum milioides (C3–C4),
and Panicum coloratum (C4) were obtained from AusPGRIS
(Australian Plant Genetic Resources Information System,
Australia). Seeds were sown in germination trays. Three-week old
seedlings were transplanted into the experimental pots. Pots were
randomly rotated within the glasshouse chambers throughout the
experiment. There were eight pots per species, [CO2], and
temperature treatment.
Three days after transplanting, a pre-weighed layer of fine stones
was added to the soil surface to minimize direct soil evaporation.
There were two control pots, which were filled with soil but had no
plants, in each room; these were used to estimate water loss by
direct soil evaporation. Pots were irrigated to 100% capacity every
1–2 d. Pot mass was recorded before watering, and masses of all
the pots were maintained at 5.5 kg after watering. Extra care was
taken to avoid overwatering and prevent water accumulation at
Downloaded from http://jxb.oxfordjournals.org/ at OUP site access on April 22, 2013
CO2 assimilation/stomatal conductance) is usually higher in
C4 than in C3 plants. Relative to C3 plants, Rubisco of C4
plants is faster (higher catalytic turnover rate) and operates
under saturating [CO2]. Thus, C4 plants typically achieve
higher photosynthetic rates with less Rubisco and leaf
nitrogen (N). Hence, leaf-level photosynthetic N-use efficiency (PNUE, rate of CO2 assimilation/leaf N concentration) is higher in C4 than in C3 plants (Brown, 1978; Taylor
et al., 2010; Ghannoum et al., 2011). Although C4 photosynthesis requires additional ATP for the regeneration of PEP,
the energy cost of photorespiration exceeds that associated
with overcycling of CO2 into the BSC at temperatures above
25 C (Ehleringer and Bjorkman, 1977).
The translation of PWUE and PNUE advantages conferred by C4 photosynthesis at the whole-plant level is less
consistent. Whole-plant WUE (plant dry mass/cumulative
water use) and NUE (plant dry mass/total leaf N) depend
on additional non-photosynthetic factors including biomass
partitioning, and shoot and root respiration (Farquhar et al.,
1989). Nevertheless, greater whole-plant WUE and NUE
have been observed in C4 relative to C3 species (Brown, 1978;
Osmond et al., 1982; Sage and Pearcy, 1987). Moreover, C3
and C4 plants have a distinct geographic distribution. C4
grasses prevail in summer-dominated rainfall regions whereas
C3 grasses prevail in winter-dominated rainfall regions. The
abundance of C4 grasses increases with increasing growing
season temperature and aridity (Hattersley, 1983; Edwards and
Still, 2008). To date, variations in photosynthetic quantum
yield provide the best theoretical framework for explaining
differences in the geographic distribution between C3 and C4
grasses (Ehleringer, 1978; Ehleringer et al., 1997). Consequently, photosynthetic characteristics have demonstrable
impacts on plant physiological and ecological functions.
A small number (20–30) of plant species have been
identified as possessing intermediate C3 and C4 photosynthetic characteristics and CO2 compensation points (Monson
and Moore, 1989; Rawsthorne, 1992; Vogan et al., 2007).
These intermediate species are probably remnants of the
complex processes that led to the evolution of C4 plants from
C3 ancestors. The small number of intermediate species
found so far raises questions about their physiological and
ecological fitness, and whether they represent living fossils of
evolutionary paths or dead-ends (Monson and Moore, 1989).
Leaves of all C3–C4 intermediates have partial or full Kranz
anatomy. Biochemically, C3–C4 intermediates differ in both
the way and the extent to which CO2 is concentrated in the
BSC (Brown, 1980; Brown et al., 1983; Ku et al., 1983, 1991;
Brown and Hattersley, 1989). For the three intermediate
Panicum species, including the one used in this study, CO2
compensation points are lowered relative to C3 leaves due to
a weak photorespiratory pump. In these species, glycine
decarboxylase activity is localized to BSCs, away from MCs.
Photorespired CO2 diffuses back through MCs, where it may
be refixed by Rubisco in MCs. In addition, this system may
weakly elevate CO2 in BSCs under high photorespiration
(Edwards and Hatch, 1982; Hylton et al., 1988; Sage, 2005).
Early work conducted with these species yielded inconclusive
evidence on the possible photosynthetic advantages of the
Resource use efficiency in C3, C3–C4, and C4 Panicum | 3225
the bottom of the pots. A commercial fertilizer (General Purpose,
Thrive Professional, Yates, Australia) was used on two occasions
(0.2 g N l1). Total, cumulative water use was calculated by
summing daily water use and subtracting the amount of water lost
by control pots without plants (Ghannoum et al., 2002).
Growth and nitrogen analyses
Plants were harvested 12 weeks after transplanting. Before harvest,
pots were weighed. At harvest, total leaf area was measured using
a leaf area meter (LI-3100A, LI-COR). The total number of leaves
and the area of one LFEL were also recorded. Shoots were
separated into stems and leaves. Roots were washed free of soil.
Plant tissues were oven-dried at 80 C for 48 h before dry mass
was measured. Leaf mass per area (LMA, g m2) was calculated as
total leaf mass (g)/total leaf area (cm2). Dried leaves of three plants
per treatment were milled to a fine powder for the determination
of N content using a CHN analyser (LECO TruSpec, LECO
Corporation, Michigan, USA).
WUE and NUE calculations
PWUE was calculated as Asat (lmol m2 s1)/gs (mol m2 s1).
Whole-plant WUE was calculated as total plant dry mass (g
plant1)/total cumulative water use of individual plants (g
plant1). PNUE was calculated as Asat (lmol m2 s1)/leaf N
concentration (mmol m2). Whole-plant NUE was calculated as
the ratio of plant dry mass (g plant1)/total leaf N at harvest (mg).
Internal leaf anatomy
To ascertain the photosynthetic type of the species used in this
study, interveinal distances and BSC arrangements were determined using light microscopy. For each species, two leaf sections
(439 mm) were cut using razor blades from either side of the leaf
mid-vein of two replicate plants. Sections were fixed in a standard
paraformaldehyde–glutaraldehyde fixative then subjected to an
ethanol dehydration series (10–100% ethanol) followed by a series
of 1:3, 1:1, and 3:1 LR White Resin:ethanol washes. Sections
were then embedded into LR White Resin (ProSci Tech, QLD,
Statistical analysis
There were five and four replicate measurements per treatment for
the growth–water use and gas exchange measurements, respectively. There were three replicate measurements for the leaf N
analysis and the A–Ci curves. Analysis of variance (ANOVA) was
conducted on data from individual plants. Data were tested for
normal distribution; extreme outliers were removed before analysis. Statistical analyses were carried out using a factorial ANOVA
(Statistica, StatSoft Inc., OK, USA) with species, growth [CO2],
and growth temperature as independent factors. Data were plotted
using Origin graphical software (Microcol Origin version 6.0,
Microcol Software, Inc., Northampton, MA, USA).
Results
Leaf anatomy, gas exchange, and chlorophyll
fluorescence
Microscopic examination of leaf sections revealed welldeveloped BSCs for P. milioides, in accordance with previous
observations (Brown and Brown, 1975; Brown et al., 1983).
Panicum bisulcatum and P. coloratum had classical C3 and C4
leaf structure (data not shown). Average interveinal distances
(mean 6SE of three sections) were 20163, 13663, and
9864 lm for P. bisulcatum, P. milioides, and P. coloratum,
respectively. These data confirmed that P. milioides has
intermediate C3–C4 leaf anatomy (Ohsugi and Murata, 1986;
Dengler et al., 1994).
When measured under current ambient [CO2] and temperature, the light-saturated photosynthetic rate (Asat) was
highest in C4, intermediate in C3–C4, and lowest in C3 leaves.
The effects of [CO2] on Asat differed between species and
growth temperature. For the C3 and C3–C4 species, increasing
[CO2] stimulated Asat similarly at both growth temperatures.
C3–C4 plants grown at subambient and ambient [CO2] had
lower Asat at high relative to ambient temperature. For the C4
species, Asat was lower at subambient [CO2] and ambient
temperature relative to the other treatments (Fig. 1A, B;
Table 1). Under current ambient [CO2] and temperature,
stomatal conductance (gs) was higher in C3 and C3–C4
relative to C4 leaves. Increasing [CO2] reduced gs in C4 plants
at both growth temperatures and in C3 plants at ambient
temperature. For C3–C4 and high temperature-grown C3
plants, gs decreased between ambient and elevated [CO2].
High temperature increased gs of the C4 species at subambient
and ambient [CO2] and of the C3 species at ambient [CO2]
(Fig. 1C, D; Table 1). Under current ambient [CO2] and
temperature, the ratio of intercellular to ambient [CO2]
(Ci/Ca) was similar between C3 and C3–C4 plants and higher
relative to C4 plants. Growth [CO2] had no significant effect
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Gas exchange measurements
Concurrent leaf gas exchange and chlorophyll fluorescence were
measured using a portable open gas exchange system (LI-6400XT,
LI-COR, Lincoln, NE, USA) connected to a leaf chamber
fluorometer (6400-40, LI-COR) to determine the light-saturated
photosynthetic rate (Asat), stomatal conductance for water vapour
(gs), ratio of intercellular to ambient [CO2] (Ci/Ca), and F’v/F’m.
These measurements were used to calculate the ratio of the quantum
yield of photosystem II (PSII) activity to the CO2 assimilation rate
(UPSII/UCO2). Measurements were taken between 10:00 h and
14:00 h on an attached last fully expanded leaf (LFEF) of the main
stem, 7 weeks after transplanting. Leaves of P. bisulcatum and
P. coloratum filled the 2 cm2 chamber; for P. milioides, leaf area was
estimated by measuring the length and width of leaf sections.
Measurements were made at a light intensity of 1800 lmol m2 s1,
leaf-to-air vapour pressure deficit of 2.3–2.8 kPa, target growth
[CO2] (280, 400, or 640 ll l1) and target mid-day growth
temperature (26 C or 30 C). Before each measurement, the leaf
was allowed to stabilize for 10–20 min until it reached a steady state
of CO2 uptake. There were four replicate measurements per
treatment. The A–Ci response curves were measured by raising
cuvette [CO2] in 10 steps (40, 70, 150, 230, 280, 400, 640, 900, 1200,
and 1800 ll l1). Relative stomatal limitation (Ls) was calculated
from A–Ci curves as Ls¼(A0–A)/A0, where A denotes the net rate of
CO2 assimilation at current ambient [CO2] and subscript zero
denotes potential A if stomatal resistance were zero (i.e. Ci;Ca)
(Farquhar and Sharkey, 1982). There were 3–4 replicate leaves
measured per species and treatment.
Australia) in polypropylene capsules and kept overnight at 80 C.
Transverse sections of 2 lm were cut using a rotary microtome
(Leica RM 2165, Leica Microsystems GmbH, Wetzlar, Germany),
heat-fixed to a microscope slide, and stained with 0.1% toluidine
blue. The slides were viewed through an Olympus compound light
microscope (Olympus BX60, Center Valley, PA, USA), and images
were collected using a connected JenOptik C14 digital camera with
130031030 resolution. Image-Pro Plus (version 5.1) was used to
determine interveinal distances. Interveinal distances represent the
averages of three observations for each leaf section.
3226 | Pinto et al.
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Fig. 1. Light-saturated rates of photosynthesis, Asat (A and B), stomatal conductance, gs (C and D), the ratio of intercellular to ambient
[CO2], Ci/Ca (E and F), and the ratio of quantum yields of PSII activity to CO2 assimilation rates, UPSII/UCO2 (G and H) of P. bisulcatum
(C3) (filled triangles), P. milioides (C3–C4) (open triangles), and P. coloratum (C4) (filled circles) grown at three atmospheric [CO2] with
a daily average of 280, 400, or 640 ll l1, and two air temperatures (ambient or ambient temperature + 4 C). Values represent the
means of four replicates 6SE. Measurements were made 7–8 weeks after planting.
on Ci/Ca, while high temperature increased the average Ci/Ca
of the Panicum species (Fig. 1E, F; Table 1). The ratio of the
quantum yields of PSII activity to CO2 assimilation rate (UPSII/
UCO2) measured under current ambient [CO2] and temperature
was higher in C3 plants relative to C4 and C3–C4 plants, which
had similar UPSII/UCO2. At ambient temperature, UPSII/UCO2
decreased with increasing [CO2] in C3 plants; at high temperature, UPSII/UCO2 decreased between subambient and ambient
Resource use efficiency in C3, C3–C4, and C4 Panicum | 3227
Table 1. Summary of three-way ANOVA for the effects of species, [CO2], and temperature on various parameters collected for
P. bisulcatum (C3), P. milioides (C3–C4), and P. coloratum (C4) grown at three atmospheric [CO2] and two air temperatures
Significance levels are NS, not significant (P >0.05); *P <0.05; **P <0.01; ***P <0.001.
Parameter
Main effects
2
1
Spp.
Temp
CO2
Spp.3Temp
Spp.3CO2
CO23Temp
Spp.3CO23Temp
***
***
***
***
***
***
***
***
***
***
NS
***
***
***
NS
NS
*
***
*
NS
***
***
***
NS
NS
NS
**
***
NS
***
***
***
NS
***
NS
***
***
NS
NS
***
**
***
***
***
**
***
NS
NS
NS
NS
***
*
***
**
***
*
NS
NS
NS
NS
**
*
***
*
NS
***
***
***
***
***
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
***
***
***
**
*
NS
NS
*
NS
*
***
*
NS
NS
NS
***
**
***
*
NS
*
NS
NS
NS
NS
[CO2] in C3 and C3–C4 plants. High temperature increased
UPSII/UCO2 of C3–C4 plants at subambient [CO2] (Fig. 1G, H;
Table 1).
The response of CO2 assimilation rate to increasing
Ci (A–Ci curve) showed a similar pattern for the C3 and
C3–C4 leaves. Gross CO2 compensation points were lower
in C4 leaves but not significantly different between C3 and
C3–C4 leaves. Elevated [CO2] reduced maximal photosynthetic rates of C3 leaves at high temperature. Panicum
coloratum showed typical C4 A–Ci curves which were not
affected by growth [CO2]. Maximal photosynthetic rates were
generally lower at high temperature (Fig. 2). Relative
stomatal limitation (Ls) was highest in C3 (39%) leaves and
not significantly different between C3–C4 (23%) and C4 (17%)
leaves under all growth conditions (Figs 2, 3). Growth [CO2]
and temperature had no effect on Ls of the Panicum species
(Table 1). There was a linear relationship between Ls and
Ci/Ca measured for the C3 and C4 leaves only; Ls of C3–C4
leaves clustered outside this relationship (Fig. 3). The C3–C4
leaves had C3-like Ci/Ca values and C4-like Ls values (Fig. 3).
Plant growth and water use
Under current ambient [CO2] and temperature, plant dry
mass and total leaf area were highest in C3, intermediate in
C4, and lowest in C3–C4 plants; total water use was similar
for C4 and C3–C4 plants but smaller relative to C3 plants
(Fig. 4, Table 1). The C3 plants also had the highest leaf area
ratio (LAR, total leaf area/plant dry mass; P <0.001; data
not shown). Under ambient temperature, increasing [CO2]
enhanced plant dry mass and leaf area in the C3 species but
not in the C3–C4 or C4 species. Under high temperature,
increasing [CO2] stimulated plant dry mass and leaf area in
the C3 and C3–C4 species; C4 plants grown at elevated [CO2]
and high temperature had greater plant dry mass and leaf
area relative to other treatments. Growth [CO2] had no
significant effect on total water used by the Panicum species.
High temperature increased plant dry mass, leaf area, and
water use of all three species (Fig. 4; Table 1). Increased leaf
area was correlated with greater leaf number rather than leaf
size (data not shown).
Leaf [N] and mass per area
At ambient temperature, LMA was highest in C3–C4 and
lowest in C3 species. At high temperature, LMA was highest
in C4 and lowest in C3 species. At elevated [CO2], LMA was
similar for all Panicum species at both growth temperatures
(Fig. 5A, B; Table 1). Under current ambient [CO2] and
temperature, leaf N concentration per unit dry mass ([N]mass)
was highest in C3–C4 relative to C3 and C4 plants. Increasing
[CO2] reduced leaf [N]mass in the C3 species. At ambient
[CO2], high temperature-grown C3 plants had higher leaf
[N]mass relative to ambient temperature (Fig. 5C, D; Table 1).
Under current ambient [CO2] and temperature, leaf N
concentration per unit leaf area ([N]area) was highest in C3–
C4 plants and lowest in C3 plants. Increasing [CO2] reduced
leaf [N]area mainly in C4 plants at high temperature. Relative
to ambient temperature, high temperature-grown plants had
lower leaf [N]area in the C3–C4 species at ambient [CO2] and
higher leaf [N]area in the C4 species at subambient [CO2]
(Fig. 5E, F; Table 1).
Leaf and whole-plant water and nitrogen use
efficiencies
When measured under current ambient [CO2] and temperature, PWUE was higher in C4 relative to C3 and C3–C4 plants,
which had similar PWUE. For the Panicum species, increasing
[CO2] stimulated PWUE at both growth temperatures. PWUE
Downloaded from http://jxb.oxfordjournals.org/ at OUP site access on April 22, 2013
Asat (lmol m s )
gs (mol m2 s1)
Ci/Ca
UPSII/UCO2
Ls (%)
Plant dry mass (g plant1)
Total leaf area (cm2 plant1)
Plant water use (kg plant1)
LMA (g m2)
Leaf N (mg g1)
Leaf N (mmol m2)
PWUE [(lmol mol H2O) 1]
Plant WUE [g (kg H2O) 1]
PNUE [mmol (mol N)1 s1]
Plant NUE [g (mg leaf N) 1]
Interactions
3228 | Pinto et al.
Fig. 2. Response of CO2 assimilation rates to increasing intercellular [CO2] in P. bisulcatum (A and B), P. milioides (C and D),
and P. coloratum (E and F) grown at three atmospheric [CO2] with
a daily average of 280 (inverted open triangles), 400 (open circles),
or 640 (filled triangles) ll l1, and two air temperatures: ambient
(A, C and E) or ambient temperature + 4 C (B, D and F). Gas
exchange measurements were made at growth temperature 7–8
weeks after planting. Values represent the means of three
replicates 6SE. Averages of relative stomatal limitation (Ls) are
shown for each species. Other details are as described for Fig. 1.
was lower at high relative to ambient temperature in the
three Panicum species (Fig. 6A, B; Table 1). Under current
ambient [CO2] and temperature, whole-plant WUE was
highest in C4, intermediate in C3, and lowest in C3–C4 plants.
Increasing [CO2] enhanced plant WUE in C3–C4 and C4
plants more than in C3 plants (P <0.1). High temperature
reduced plant WUE of the Panicum species mainly at
elevated [CO2] (Fig. 6C, D: Table 1).
When measured under current ambient [CO2] and temperature, PNUE was lower in C3–C4 relative to C3 and C4
plants, which had similar PNUE. Increasing [CO2] enhanced PNUE of the three Panicum species at both growth
temperatures. High temperature had no significant effect on
PNUE of the three species (Fig. 7A, B; Table 1). Under
current ambient [CO2] and temperature, plant NUE was
similar between the three Panicum species. Under ambient
temperature, plant NUE increased between ambient and
elevated [CO2]. Under high temperature, plant NUE increased
from subambient to ambient [CO2] in the C3–C4 and C4 plants
Fig. 4. Plant dry mass (A and B), total leaf area (C and D), and
cumulative plant water use (E and F) of P. bisulcatum, P. milioides,
and P. coloratum grown at three atmospheric [CO2] and two air
temperatures. Plants were harvested 12 weeks after transplanting.
Values represent the means of five replicates 6SE. Other details
are as described for Fig. 1.
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Fig. 3. Relationship between relative stomatal limitation (Ls)
calculated from the A–Ci curves (shown in Fig. 2) and Ci/Ca
measured at growth [CO2] and temperature (shown in Fig. 1) for
P. bisulcatum, P. milioides, and P. coloratum grown at three
atmospheric [CO2] and two air temperatures. The solid line
represents the linear regression of data points for the C3 and C4
species only (excluding values for the C3–C4 species—encircled
within the dashed line). Other details are as described for Fig. 1.
Resource use efficiency in C3, C3–C4, and C4 Panicum | 3229
Fig. 5. Leaf mass per area, LMA (A and B), and leaf N per unit leaf
dry mass (C and D) and unit leaf area (E and F) of P. bisulcatum,
P. milioides, and P. coloratum grown at three atmospheric [CO2]
and two air temperatures. Other details are as described for Fig. 1.
and between ambient and elevated [CO2] in the C3 plants. High
temperature increased plant NUE of the Panicum species at
ambient and elevated [CO2] (Fig. 7C, D; Table 1).
Leaf- and plant-level WUE measured in the three species
and under the six growth conditions were linearly related;
PWUE explained 60% of the variation in plant WUE
(Fig. 8A). No similar relationship was observed between
PNUE and plant NUE (Fig. 8B). There was a strong,
positive relationship between PWUE and PNUE measured in this study (Fig. 8C).
Discussion
Photosynthesis and growth of the C3, C3–C4,
and C4 Panicum species
Fig. 6. Photosynthetic WUE (PWUE; A and B), and whole-plant
WUE (C and D) of P. bisulcatum, P. milioides, and P. coloratum
grown at three atmospheric [CO2] and two air temperatures. Other
details are as described for Fig. 1.
Under current ambient [CO2] and temperature, P. milioides
(C3–C4) had intermediate Asat relative to the C3 and C4
Panicum species (Fig. 1A, B). Interestingly, C3–C4 photosynthesis reduced the overall stomatal limitation relative to C3
photosynthesis without affecting Ci/Ca, as otherwise is the
case for C4 photosynthesis (Fig. 3). Lower stomatal limitation may be due to reduced CO2 limitation as a result of
partial recycling of photorespired CO2 during C3–C4 photosynthesis (Brown et al., 1991). The C3–C4 photosynthetic
advantage diminished under elevated [CO2]. Hence, relative
to C3 photosynthesis, the C3–C4 pathway did not boost
photosynthetic capacity but rather alleviated stomatal limitation (Figs 2, 3). The UPSII/UCO2 ratio provides an estimate of
the photosynthetic electron cost. Under most conditions,
UPSII/UCO2 was similar between the C3–C4 and C4 species
except for subambient [CO2] and high temperature, where
UPSII/UCO2 was similar between C3 and C3–C4 plants
(Fig. 1G, H). These results suggest that the ratio of Rubisco
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Fig. 7. Photosynthetic NUE (PNUE; A and B) and whole-plant
NUE (C and D) of P. bisulcatum, P. milioides, and P. coloratum
grown at three atmospheric [CO2] and two air temperatures. Other
details are as described for Fig. 1.
3230 | Pinto et al.
oxygenation to carboxylation was generally lower in C3–C4
relative to C3 plants. This can be attributed to a lower [O2]/
[CO2] ratio, i.e. a marginal CO2-concentrating mechanism, in
BSCs of C3–C4 leaves relative to MCs of C3 leaves. A lower
[O2]/[CO2] ratio may result from the local delivery of CO2
by glycine decarboxylation (Hylton et al., 1988) or lower
PSII activity in BSC chloroplasts of C3–C4 intermediates
as usually observed in BSC chloroplasts of C4 species
Photosynthetic WUE in C3, C3–C4, and C4 Panicum
species
In this study, C3–C4 and C3 leaves did not have significantly
different PWUE (Fig. 6A, B). During both C3 and C3–C4
Downloaded from http://jxb.oxfordjournals.org/ at OUP site access on April 22, 2013
Fig. 8. Relationships between leaf- and plant-level WUE (A) and NUE
(B) and between PNUE and PWUE (C) in P. bisulcatum, P. milioides,
and P. coloratum grown at three atmospheric [CO2] and two air
temperatures. The solid lines in A and C represent linear regressions
of all data points. Other details are as described for Fig. 1.
(Ghannoum et al., 2005). The latter aspect has not been
investigated in C3–C4 intermediates. Under subambient
[CO2] and high temperature, UPSII/UCO2 was similar between
C3 and C3–C4 leaves, indicating that with increasing photorespiration, the proportion of CO2 that cannot be refixed in
the C3–C4 leaves increases.
For the C3 species, increasing [CO2] stimulated Asat and
reduced energy partitioning to photorespiration as indicated
by the similar UPSII/UCO2 between C3 and C4 leaves at
elevated [CO2]. These responses are commonly reported for C3
plants exposed to elevated [CO2] (Ainsworth and Rogers,
2007). As expected, Asat showed a weak CO2 response while
UPSII/UCO2 did not change with increasing [CO2] in the C4
species (Ghannoum et al., 2000). At high temperature, Asat
was either unaffected (C3 and C4) or reduced (C3–C4) in the
Panicum species, indicating strong thermal acclimation of
photosynthesis. Most plant species acclimate to changes in
growth temperature by shifting the photosynthetic thermal
optimum closer to the new growth conditions (Berry and
Björkman, 1980; Dwyer et al., 2007; Sage and Kubien, 2007;
Ghannoum et al., 2010b). Reduced Asat at high temperature in
P. milioides may reflect lower photosynthetic thermal optima
in this C3–C4 species relative to the C3 and C4 congeners.
Whole-plant biomass accumulation can provide a longterm integrative response of the instantaneous photosynthetic
responses to growth [CO2]. In line with the photosynthetic
responses and the published literature, the C3 Panicum species
showed a consistent biomass response to increasing growth
[CO2] from subambient to elevated [CO2] (Ghannoum et al.,
1997, 2010a; Wand et al., 1999). Under high temperature, the
C3–C4 plants had a similar growth responsiveness, and hence
CO2 limitation, to their C3 counterparts. Under ambient
temperature, the growth response of C3–C4 plants to increasing [CO2] was weak (Fig. 4A, B). These results indirectly
suggest that CO2 recycling during C3–C4 photosynthesis
improves the carbon budget of C3–C4 plants, thus reducing
their CO2 limitation. The relative significance of CO2
recycling diminishes as photorespiration increases (e.g. due
to high temperature) beyond the capacity of BSCs and MCs
to refix increasing amounts of photorespired CO2. In contrast
to previous interpretations (Monson and Moore, 1989;
Schuster and Monson, 1990), the present results (plant dry
mass; UPSII/UCO2) imply that, in relative terms, the contribution of CO2 recycling to CO2 sequestration by C3–C4 plants is
reduced at low [CO2] and high temperature, i.e. with
increasing photorespiration. C4 plants accumulated more dry
mass under elevated [CO2] and high temperature only, most
probably due to reduced gs, and hence plant water use. This
effect was more pronounced in the larger elevated [CO2]- and
high temperature-grown plants because they are more likely
to reduce soil moisture substantially during the course of the
day (Ghannoum et al., 2000; Seneweera et al., 2001).
Resource use efficiency in C3, C3–C4, and C4 Panicum | 3231
et al. (2005) demonstrated that Asat per Rubisco explains
most of the variation in PNUE amongst C4 grasses. Higher
Asat per Rubisco is also expected to enhance PNUE in C4
relative to C3 leaves. While this has been often observed
(Taylor et al., 2010), it was not the case in the current study.
Panicum coloratum is a forage grass species which may
explain its unusually higher leaf [N] relative to other C3 and
C4 grasses (Ghannoum et al., 1997; Ghannoum and Conroy,
1998). Panicum milioides (C3–C4) had the highest leaf [N] and
lowest PNUE in the current study. High leaf [N] may reflect
higher photosynthetic or non-photosynthetic N investment.
In our study, carboxylation efficiency, as determined from
the initial slope of the A–Ci response curves, was similar for
C3 and C3–C4 leaves. Similar carboxylation efficiencies and
Rubisco activities were reported between P. bisulcatum (C3)
and P. milioides in an early study (Ku and Edwards, 1978).
These results suggest that C3 and C3–C4 species had similar
Rubisco activity per leaf area. Hence, it can be concluded
that P. milioides had higher Asat per Rubisco despite its lower
PNUE relative to the C3 Panicum species. Lower PNUE and
higher leaf [N] in P. milioides may reflect the greater nonphotosynthetic N requirements of the C3–C4 pathway, such
as the N construction costs associated with the provision of
BSCs in C3–C4 intermediates (Schuster and Monson, 1990).
The trends in PNUE observed here need to be confirmed
using more phylogenetically related species.
Increasing [CO2] stimulated PNUE mainly by enhancing
Asat of C3 and C3–C4 plants, and reducing leaf [N]area of C4
plants. Reduced leaf [N] is a commonly observed response
in C3 plants to growth at elevated [CO2], but there are
frequent exceptions especially in vegetative seedlings supplied with non-limiting nutrients (Conroy and Hocking,
1993; Ainsworth and Rogers, 2007; Ghannoum et al., 2010b).
For C4 plants, reduced leaf [N] at elevated [CO2] has been
reported in some studies (LeCain and Morgan, 1998) but not
in others (Ghannoum et al., 1997). In the present study,
reduced leaf [N]area in the C4 species was due to lower LMA,
possibly as a result of improved water relations at elevated
[CO2] (Ghannoum et al., 2000; Poorter et al., 2009). High
temperature had no significant effect on PNUE, given that
both Asat and leaf [N] were unchanged or slightly reduced at
high temperature.
Photosynthetic NUE in C3, C3–C4, and C4 Panicum
species
Leaf-level WUE and NUE have received more attention
than the corresponding plant-level traits. It can be argued
that both types of parameters, as measured in the present
study, represent snapshots at a particular time of the day or
developmental stage. Nevertheless, plant-level WUE and
NUE provide longer term and more integrative estimates
than those measured at the leaf level, and may be more
relevant to plant productivity and survival in the natural
environment. Measuring plant WUE and NUE can also
reveal the extent to which photosynthetic efficiencies determine whole plant characteristics. For the Panicum species
used in this study, there was a more consistent relationship
In contrast to PWUE, variations in PNUE were not solely
predictable based on the photosynthetic pathway (Fig. 7A, B).
It is well established that relative to C3, C4 leaves
operate with higher carboxylation efficiency due to the
higher activity of PEPC relative to Rubisco and the CO2
saturation of Rubisco. This, together with the higher
catalytic turnover rate of C4 relative to C3 Rubisco, often
leads to higher Asat per Rubisco active sites (Long, 1999;
von Caemmerer and Quick, 2000; Sage, 2002). Ghannoum
Whole-plant WUE and NUE in C3, C3–C4, and C4
Panicum species
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photosynthesis, delivery of CO2 to Rubisco in the MC relies
on diffusion down a narrow concentration gradient which
necessitates greater stomatal opening relative to C4 leaves.
On average, gs, Ci/Ca, and the responsiveness of photosynthetic rates to [CO2] were similar between C3 and C3–C4
plants. Hence, C3–C4 photosynthesis in P. milioides
offered no PWUE advantage over C3 photosynthesis in
P. bisulcatum, even at subambient [CO2]. These results are
in line with an early study using P. milioides (Brown and
Simmons, 1979) but not with another (Ku and Edwards,
1978). In contrast, the C4 species P. coloratum maintained
higher PWUE than its C3 and C3–C4 counterparts under all
growth conditions. Improved PWUE is one of the most
consistently reported advantages of C4 photosynthesis. The
CO2-concentrating mechanism saturates Rubisco with CO2
at a lower Ci relative to C3 species, allowing C4 leaves to
operate with lower gs relative to C3 leaves (Long, 1999;
Taylor et al., 2010; Ghannoum et al., 2011). Results obtained
using the more widely studied Flaveria (dicot) intermediate
species indicated that C4-like PWUE depends on the
establishment of significant C4 cycle activity and a nearcomplete localization of the C3 and C4 cycle carboxylases
and decarboxylases (Monson, 1989; Monson and Moore,
1989; Schuster and Monson, 1990; Apel, 1994). Accordingly,
leaky C4-like CO2-concentrating systems (e.g. Flaveria spp.)
and CO2 refixation (e.g. Flaveria and Panicum spp.) may
improve photosynthetic rates and reduce photorespiration
relative to related C3 species; however, these advantages do
not necessarily improve PWUE to the same extent as in C4
species with fully developed CO2-concentrating mechanisms.
Increasing [CO2] stimulated PWUE of all the three
grasses similarly but not by the same mechanisms. In the
C4 species, increasing [CO2] stimulated PWUE mainly by
reducing gs. In the C3 and C3–C4 species, increasing [CO2]
stimulated PWUE by enhancing Asat and reducing gs. These
results demonstrate that gs was more sensitive to increasing
[CO2] in C4 relative to C3 and C3–C4 leaves. In agreement,
Huxman and Monson (2003) showed that the slope of the
relationship between gs and Ci was steeper in C4 than in C3
and C3–C4 intermediate Flaveria species. However, Morison
and Gifford (1983) found no evidence that stomata were more
sensitive to [CO2] in C4 relative to C3 grasses. More work is
needed to resolve this aspect of the CO2 response. High
temperature reduced PWUE of the Panicum species mainly by
increasing gs and, in some instances, decreasing Asat.
3232 | Pinto et al.
Conclusions
Results obtained in this study using Panicum intermediates
and those previously reported using other types of C3–C4
intermediates demonstrate that, short of substantial C4
cycle activity and advanced cell-specific localization of C3
and C4 cycle enzymes between the MC and BSC, photosynthetic intermediacy does not improve WUE. In addition, it
may reduce NUE at both the leaf and plant levels and
under a wide range of growth conditions. Partial recycling
of photorespired CO2 may reduce energy loss and stomatal
limitation normally associated with C3 photosynthesis.
Improvement in plant productivity, however, is also contingent on the species’ intrinsic relative growth rate. Overall,
there was a positive relationship between PWUE and
PNUE across species and growth conditions. However,
only PWUE clearly translated into improved plant WUE.
This may highlight the strong ecological nature of plant
NUE. The type of C3–C4 photosynthesis present in Panicum
species reflects early evolutionary changes on the path from
C3 towards C4 photosynthesis; however, it does not present
ecophysiological advantages under climatic scenarios of the
recent past and future.
Acknowledgements
We thank Anthony Newton and Liz Kabanoff for assistance with light microscopy, and Jann Conroy for critically
reading this manuscript. HP was supported by a combined
Summer–Honours scholarship from UWS. This research
was also supported by funding from the Australian Research Council (DP0879531) to DTT.
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