Journal of Experimental Botany, Vol. 59, No. 7, pp. 1705–1714, 2008
doi:10.1093/jxb/erm210 Advance Access publication 7 February, 2008
SPECIAL ISSUE RESEARCH PAPER
Consequences of C4 photosynthesis for the partitioning of
growth: a test using C3 and C4 subspecies of Alloteropsis
semialata under nitrogen-limitation
Brad S. Ripley1,*, Trevor I. Abraham1 and Colin P. Osborne2
1
2
Botany Department, Rhodes University, PO Box 94, Grahamstown 6140, South Africa
Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK
Received 10 May 2007; Revised 6 August 2007; Accepted 7 August 2007
Abstract
Introduction
C4 plants dominate the world’s subtropical grasslands,
but investigations of their ecology typically focus on
climatic variation, ignoring correlated changes in soil
nutrient concentration. The hypothesis that higher
photosynthetic nitrogen use efficiency (PNUE) in C4
than in C3 species allows greater flexibility in the
partitioning of growth, especially under nutrientdeficient conditions, is tested here. Our experiment
applied three levels of N supply to the subtropical
grass Alloteropsis semialata, a unique model system
with C3 and C4 subspecies. Photosynthesis was
significantly higher for the same investment of leaf N
in the C4 than C3 subspecies, and was unaffected by N
treatments. The C4 plants produced more biomass
than the C3 plants at high N levels, diverting a greater
fraction of growth into inflorescences and corms, but
less into roots and leaves. However, N-limitation of
biomass production caused size-dependent shifts in
the partitioning of growth. Root production was higher
in small than large plants, and associated with decreasing leaf biomass in the C3, and inflorescence
production in the C4 plants. Higher PNUE in the C4
than C3 subspecies was therefore linked with greater
investment in sexual reproduction and storage, and
the avoidance of N-limitations on leaf growth, suggesting advantages of the C4 pathway in disturbed and
infertile ecosystems.
Plants using the C4 photosynthetic pathway dominate
subtropical grasslands covering approximately 13% of the
Earth’s land surface (Still et al., 2003). Most studies
investigating the ecological significance of this pathway
have focused on plant responses to atmospheric CO2 and
climatic variables (e.g. Owensby et al., 1996; Kubien and
Sage, 2004). However, soil nitrogen (N) availability is
a limiting factor for plant growth in many terrestrial
ecosystems, particularly in the tropical regions (Field and
Mooney, 1986), and varies significantly along climatic
gradients (Scholes, 1990; Drury et al., 2003; Paul et al.,
2003). A mechanistic understanding of how N-use varies
among plant species with different photosynthetic pathways is therefore critical for interpreting spatial and
temporal variation in grassland productivity and community composition.
In C3 plants, the photosynthetic enzyme Rubisco
accounts for up to 30% of the leaf N content (Lawlor
et al., 1989), but accounts for only 4–21% of leaf N in C4
species (Sage et al., 1987; Evans and von Caemmerer,
2000; Lawlor et al., 2001). The lower N requirement of
C4 plants results from their CO2-concentrating mechanism
(CCM), which raises the bundle sheath CO2 concentration, saturating Rubisco and almost eliminating photorespiration. Without this mechanism, Rubisco in the C3
photosynthetic pathway operates at only 25% of its
capacity (Sage et al., 1987) and loses c. 25% of fixed
carbon to photorespiration (Ludwig and Canvin, 1971).
To attain comparable photosynthetic rates to those in C4
plants, C3 leaves must therefore invest more heavily in
Rubisco and have a greater N requirement. Because the
Key words: Alloteropsis semialata, biomass partitioning, C4
photosynthesis, nitrogen-limitation, photosynthetic nitrogen use
efficiency.
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: Rubisco, ribulose-1,5-bisphosphate carboxylase oxygenase; PEPc, phosphoenolpyruvate carboxylase; CCM, CO2- concentrating
mechanism; PNUE, photosynthetic nitrogen use efficiency.
ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1706 Ripley et al.
Rubisco specificity for CO2 decreases with increasing
temperature (Long, 1991), this difference between the C3
and C4 photosynthetic N-use efficiency (PNUE) is greatest
at high temperatures (Long, 1999). The high PNUE of C4
plants is partially offset by the N-requirement for CCM
enzymes, but the high maximum catalytic rate of PEPc
means that these account for only c. 5% of leaf nitrogen
(Long, 1999).
Based on these physiological differences, Long (1999)
proposed two strategies whereby C4 plants growing in
their natural environment may exploit their PNUE
advantage over C3 species. First, C4 plants could produce
a greater leaf area for the same investment of N,
increasing their ability to compete for light in productive
plant communities. In combination with higher photosynthetic rates, this increases the potential for whole-plant
photosynthetic productivity. Alternatively, C4 plants could
produce comparable leaf areas or photosynthetic rates to
C3 plants and allocate the saved N to root growth, increasing their ability to forage for soil resources, a strategy
that may be advantageous in nutrient-limited habitats. A
third strategy could favour N partitioning to reproductive
or storage organs, increasing the competitive advantage
of C4 plants in communities frequently defoliated by
disturbances such as fire or grazing, or where high
leaf mortality occurs in response to climatic extremes
such as drought or frost. The partitioning of N to
reproductive propagules is critical to regeneration after
disturbance.
The particular ecological strategy adopted is likely to be
influenced by the N-environment to which plants are
adapted, because species occupying eutrophic or mesotrophic habitats respond differently to the same N supply
treatments (Aerts and Chapin, 2000). Comparisons of the
N-use strategies in C3 and C4 plants must therefore be
made between species adapted to the same N-environment, and at ecologically relevant nutrient levels (Sage
and Pearcy, 1987). PNUE also differs between C4
photosynthetic subtypes, and this has been attributed to
differences in enzymatic requirements (Bowman, 1991;
Ghannoum et al., 2005). However, in the grass family,
such findings are confounded by phylogenetic history,
because the majority of the NAD-ME species are in the
subfamily Chloridoideae, whereas most NADP-ME
grasses are in the Panicoideae (Ellis, 1974; Ellis et al.,
1980). This highlights the need for phylogenetic control in
comparative studies to isolate the direct effects of the
photosynthetic mechanism from those of other ancestral
characteristics.
Here, the role of the photosynthetic pathway in PNUE
and growth responses to N was investigated using experiments with a unique model system; the subtropical
perennial grass Alloteropsis semialata. Alloteropsis semialata is the only species yet discovered with C3 and C4
subspecies (Gibbs Russell, 1983), and is therefore an ideal
model for investigating the interaction of photosynthetic
pathway with ecological strategy, avoiding the confounding effects of phylogenetic distance usually encountered in
comparisons of C3 and C4 species. A recent phylogenetic
analysis suggests that the C3 subspecies represents a reversion from the C4 photosynthetic type (Ibrahim et al.,
2007). Both subspecies co-occur naturally in South Africa
and grow in habitats with impoverished soils, suggesting
adaptation to a similar N-environment, and in regions
experiencing high summer temperatures (Ellis, 1974),
conditions likely to emphasize the difference in PNUE
between C3 and C4 plants. Two alternative hypotheses
were tested that: (i) the C4 produces a greater leaf area
than the C3 subspecies and, with a higher photosynthetic
rate, is more productive; or (ii) the C4 produces a similar
or lower leaf area relative to the C3 subspecies, but invests
more resources in root or reproductive growth. Another
hypothesis (iii) that, under a restricted N supply, increased
relative investment of growth in roots at the expense of
shoots is greater in the C3 than C4 subspecies was also
tested, and whether this is mediated by plant size, or is
a direct response to nutrient supply.
Materials and methods
Plant material and nutrient treatments
Established plants of the C4 subspecies Alloteropsis semialata
(R.Br.) Hitchc. subsp. semialata and C3 subspecies Alloteropsis
semialata (R.Br.) Hitchc. subsp. eckloniana (Nees) Gibbs Russell
were collected from grasslands in the surrounds of Middelburg (near
Pretoria, South Africa) and Grahamstown (near Port Elizabeth,
Eastern Cape, South Africa), respectively. These were used to
produce a collection of individually potted plants in 20 l pots, each
established from three tillers originating from an individual plant.
The plants were grown in washed river-sand, and subjected to three
levels of N application, each replicated in 6–8 pots. The N
treatments were applied by frequent watering with Long Ashton’s
solution, with any seepage from the pots collected in trays and reapplied. Different nitrogen concentrations were chosen to be
representative of, or below, levels found in savanna soils (Scholes
and Walker, 1993). Nitrogen was supplied as nitrate in the form of
KNO3 and Ca(NO3)2. The levels chosen were: 7 g N m2 year1
(High N), representative of nutrient-rich savannah soils; 3.5 g N
m2 year1 (Medium N) representative of nutrient-poor savannah
soils; and 0 g N m2 year1 (Low N), where plants depended solely
on N that remained in the sand after washing. Pots were established
and maintained in a clear polythene tunnel, where average day/
night temperatures were 31/17 C, and humidity ranged between
32% and 48%.
Biomass, leaf area, and tiller number
The plants were harvested after 23 weeks of nutrient treatment and
the sand was washed from the roots. Tillers were counted and
a subsample of three tillers from each plant used to determine leaf
area with image analysis software (WinDIAS, Delta-T Devices,
Cambridge, UK). Leaves were oven dried and weighed for the
calculation of specific leaf area (SLA). The remaining tillers from
each pot were separated into leaf laminae, leaf sheaths, culms and
inflorescences (flowers), roots and corms; the latter defined as the
Nitrogen-limitation in C3 and C4 plants 1707
below-ground portion of the swollen stem and leaf bases. These
plant parts were then oven dried to constant weight at 60 C. Linear
relationships between leaf area and weight were established from
the subsample of three tillers from each subspecies and treatment.
Slopes from these relationships had r2 >0.9 and were used to
estimate canopy leaf area from the total leaf weight for each plant.
Specific leaf area (SLA) and leaf area ratio (LAR) were calculated
according to Evans (1972).
Leaf photosynthesis and nitrogen content
After 12 weeks of treatment, CO2-responses of photosynthesis (A–
Ci curves) were measured using a photosynthesis system (LI-6400
Li-Cor Inc., Lincoln, Nebraska, USA) following Long and
Bernacchi (2003). Measurements were made on the youngest
mature leaf at 21% O2, with environmental conditions in the cuvette
set to provide a photosynthetically active photon flux density
(PPFD) of 2000 lmol m2 s1, an average leaf temperature of
26 C, and VPD <1.6 kPa. At the end of each measurement the leaf
was harvested, oven dried to constant mass at 60 C, and ground to
a homogeneous powder using a ball mill (800M mixer/mill, Glen
Creston, Middlesex, UK). Nitrogen contents were determined using
a mass spectrometer (PDZ Europa 20–20, Cheshire, UK). The
measurements were replicated on five plants from each treatment.
Photosynthetic rate and intercellular CO2 concentration (Ci) were
calculated according to von Caemmerer and Farquhar (1981) and
individual A–Ci responses fitted with the monomolecular equation:
A ¼ að1 ebc3Ci Þ, with r2 >0.97. Fitted equations were used to
interpolate data to a common series of Ci values for the calculation
of means and standard errors, and parameters a, b, and c to calculate
the CO2 compensation point (C¼b/c), carboxylation efficiency
(CE¼aceb), and CO2-saturated photosynthetic rates (AMAX¼a).
PNUE was calculated as A (measured in an atmosphere of 370 lmol
CO2 mol1) per unit leaf nitrogen.
Statistics
All parameters, with the exception of the percentage biomass,
were compared between subspecies and treatments using factorial
ANOVA (Statistica 7.0, Statsoft, Inc, Tulsa, USA). The percentage biomass parameters were compared using a Generalized
Linear Model (GLM) for data with quasibinomial distributions
(R Development Core Team, 2006).
Biomass partitioning data were also analysed as a function of
plant size with linear fits describing the resultant allometric
trajectories. Parameters were compared between subspecies using
GLM (Statistica 7.0, Statsoft, Inc, Tulsa, USA), with plant size as
a continuous predictor in order to ascertain whether allometic
trajectories were different between subspecies. As data were from
a single harvest and plant masses from each treatment only partially
overlapped, the between-treatment interactions were not included in
the statistical analysis (Poorter and Nagel, 2000).
Results
Photosynthesis and leaf nitrogen
Photosynthetic rates at the ambient atmospheric CO2
concentration of 370 lmol mol1 were significantly
higher in the C4 than C3 subspecies, but not significantly
affected by nitrogen supply (Fig. 1a; Table 1). Leaf N
content was significantly affected by nitrogen supply
when expressed as a percentage of dry weight, but not on
Fig. 1. (a) Net photosynthetic CO2-fixation (A), (b) percentage leaf
nitrogen (circles) or leaf nitrogen concentration (diamonds), and
(c) photosynthetic nitrogen use efficiency (PNUE), for C3 (closed
symbols) and C4 (open symbols) subspecies of Alloteropsis semialata
supplied the indicated levels of N. All are mean values with vertical
bars representing standard errors (n >5).
a leaf area basis. However, leaf N was not significantly
different between subspecies, irrespective of the basis of
expression (Fig. 1b; Table 1). This, in combination with
the observed effects on A, resulted in a relatively uniform
PNUE across N treatments, that was higher in the C4 than
C3 subspecies (Fig. 1c; Table 1).
A–Ci curves (Fig. 2; Table 2) showed that CE was
higher in the C4 than the C3 leaves and declined
significantly with reduced N supply, but the interaction
between subspecies and treatment was not significant
(Table 1). Although the CE was affected by N supply in
the C4 subspecies, this did not change A at the ambient
atmospheric CO2 concentrations of 370 lmol mol1
(Fig. 1a). C was higher in the C3 than the C4 plants, but
not altered by N supply (Tables 1, 2). CO2-saturated
photosynthetic rate (AMAX) was not significantly different
between the subspecies, but was decreased in the
C3 plants when the N supply was reduced below 3.5 g
N m2 year1 (Tables 1, 2).
1708 Ripley et al.
Table 1. Factorial ANOVA or GLM results for photosynthetic and biomass parameters that were compared between subspecies and
N supply treatments
Parameter
Subspecies
Treatment
Interaction
Photosynthetic rate at Ca ¼ 370 ppm (A)
Percentage nitrogen content (N%)
Nitrogen content (N g m2)
Photosynthetic N-use efficiency (PNUE)
Carboxylation efficiency (CE)
CO2 compensation point (G)
CO2-saturated photosynthetic rate (AMAX)
Whole plant biomass
Number of tillers
Root:shoot biomass
Leaf:root biomass
Percentage leaf biomass
Percentage sheath biomass
Percentage root biomass
Percentage corm biomass
Percentage flower biomass
Leaf area
Specific leaf area (SLA)
Leaf area ratio (LAR)
F¼19.4, P <0.0002
n.s.
n.s.
F¼9.1, p¼ 0.0064
F¼63.4, P <0.0001
F¼421.0, P <0.0001
n.s.
F¼17.01, P <0.0002
F¼38.8, P <0.0001
n.s.
F¼9.3, P <0.0035
F¼87.1, P <0.001
n.s.
F¼54.4, P <0.001
F¼48.4, P <0.001
F¼114.6, P <0.001
F¼11.1, P <0.002
F¼58.8, P <0.0001
F¼115.7, P <0.0001
n.s.
F¼10.9, P <0.004
n.s.
n.s.
F¼4.0, P <0.03
n.s.
F¼4.5, P <0.022
F¼63.3, P <0.0001
F¼30.9, P <0.0001
F¼25.04, P <0.0001
F¼15.1, P <0.0001
F¼8.5, P <0.001
F¼23.7, P <0.001
F¼27.2, P <0.001
n.s.
F¼3.6, P¼0.038
F¼64.8, P <0.0001
F¼13.5, P <0.0001
F¼8.7, P <0.0008
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
F¼8.6,
n.s.
n.s.
F¼4.5,
n.s.
n.s.
n.s.
n.s.
F¼4.5,
n.s.
F¼4.7,
Biomass
Although photosynthetic rates and PNUE were little
affected by the nutrient treatments, biomass was significantly reduced by a restricted N supply, and significantly
higher in the C4 than C3 plants (Fig. 3a; Table 1). The
relationship was not in direct proportion to N supply, with
an increase from 0 to 3.5 g m2 year1 having a greater
effect than that from 3.5 to 7 g m2 year1. The number
of tillers produced also differed between subspecies and
was significantly reduced at low N supply (Fig. 3b; Table
1). The C3 produced more tillers than the C4 subspecies at
medium and high levels of N supply, but not at low levels
of N supply. The reduction in tillering when N supply was
reduced from the medium- to low-level was significant in
both subspecies, but the effect was larger in the C3 plants
(Fig. 3b), resulting in a significant subspecies by treatment
interaction (Table 1).
The apportioning of this biomass between above- and
below-ground plant parts was not different between
subspecies, but root allocation was favoured at reduced
N supply (Fig. 3c; Table 1). This was also evident in the
leaf-to-root ratio which was lower in the C4 than the C3
subspecies (Table 1), and declined at low N supply
(Fig. 3d). Collectively these responses suggest a functional
shift in the relationship between nutrient acquisition and
light interception as N became limiting.
C3 plants allocated more biomass to roots than the C4
plants and both subspecies increased root allocation as N
supply was reduced (Fig. 4a; Table 1). However, the
resultant shift in growth partitioning differed between the
C3 and C4 subspecies. The C3 plants allocated a greater
proportion of biomass to leaves than the C4 plants, and
decreasing N supply reduced this percentage in the C3 but
P <0.001
P¼0.018
P <0.017
P <0.016
Fig. 2. Responses of net photosynthetic CO2-fixation (A) to intercellular
CO2 concentration (Ci) for C3 and C4 subspecies of Alloteropsis semialata.
Measurements were made on leaves of plants supplied with 7 (squares),
3 (circles), or 0 (diamonds) g N m2 year1 nitrogen. All are mean values
of interpolated data with vertical bars representing standard errors (n¼5).
Larger solid points represent values for measurements made at an ambient
atmospheric CO2 concentration of 370 lmol mol1.
not the C4 plants (Fig. 4b; Table 1). Increased root
production in the low N treatments was therefore
accompanied by decreased leaf growth in this subspecies.
By contrast, greater root production in the C4 subspecies
Nitrogen-limitation in C3 and C4 plants 1709
was tied to a reduction in reproductive growth. The C4
plants allocated more biomass to flowering than the C3
plants, and in both subspecies this allocation was reduced
when N supply was restricted (Fig. 4c; Table 1).
However, this effect of N treatment on flower allocation
only had a biologically significant effect in the C4 plants,
where 95% of individuals flowered, in contrast to the C3
plants where only 9% flowered. Unlike differences in leaf
and flower allocation, both subspecies allocated a similar
proportion of biomass to leaf sheaths, and this was
reduced when N supply was restricted (Fig. 4d; Table 1).
A further difference was observed in the allocation of
Table 2. Photosynthetic characterization of Alloteropsis semialata subspecies
Mean 6SE (n >5) are shown for the leaf CO2 compensation point (C),
carboxylation efficiency (CE), and CO2-saturated photosynthetic rate
(AMAX) of C3 and C4 subspecies supplied the indicated levels of
nitrogen. Results of ANOVA comparisons are given in Table 1.
SubspeciesN application CE
G
AMAX
(g m2 year1)(mmol m2 s1)(lmol mol1)(lmol m2 s1)
C3
C4
7.0
3.5
0.0
7.0
3.5
0.0
120620
130620
110610
390640
350650
230630
52.664.2
44.862.7
55.461.7
8.061.8
8.861.6
4.562.7
26.062.8
32.061.1
19.862.3
23.564.4
24.562.2
21.061.5
growth to corms, which was significantly greater in the C4
than the C3 species in all N treatments, but unaltered by N
supply (Fig. 4d; Table 1).
Allometric analyses
All components of plant biomass, with the exception of
flower mass and number in the C3 subtype, scaled in
direct proportion to total plant biomass (Fig. 5a–f). In
the case of leaf, root, corm, and total inflorescence mass,
and flower number, the allometric relationships were
significantly different between the C3 and C4 subspecies
(Table 3). For the vegetative components, at least 79% of
the variation within each photosynthetic subtype was
explained by total plant mass, and the adherence of these
data to single straight-line relationships, irrespective of
N supply, is consistent with subspecies-specific allometric
trajectories that were unaltered by treatment. Nutrient
treatments (indicated by different symbols in Fig. 5)
simply shifted the position of parameters along these
trajectories, with 3.5 and 7 g m2 year1 treatments
always resulting in higher total plant biomass than the 0 g
m2 s1 treatments.
Average flower mass showed the weakest allometric
relationship for the C4 subspecies (Fig. 5e). Apparently,
when plants flowered, inflorescences of similar mass were
produced, and it was the number of these on each plant
that was affected by plant allometry/nutrient treatment.
Fig. 3. (a) Whole plant dry biomass, (b) number of tillers, (c) root:shoot ratio, and (d) leaf:root biomass for C3 (closed symbols) and C4 (open
symbols) subspecies of Alloteropsis semialata supplied the indicated levels of N. Above-ground biomass includes leaf lamina, sheath, and
inflorescence, whilst below-ground biomass incorporates root and corm. All are mean values with vertical bars representing standard errors (n >6).
1710 Ripley et al.
Fig. 4. (a) Root, (b) leaf lamina, (c) flower, and (d) leaf sheath (diamonds) or corm (circles), relative biomass for C3 (closed symbols) and C4 (open
symbols) subspecies of Alloteropsis semialata supplied the indicated levels of N. All are mean values with vertical bars representing standard errors
(n >6).
Leaf area
Canopy leaf area was significantly higher in the C3 than
the C4 subtype, but was reduced at low N supply to
a greater extent in the C3 plants (Fig. 6a; Table 1).
Consequently, at the lowest level of N supply, the leaf
areas did not differ significantly between subspecies. The
specific leaf area (SLA) and leaf area ratio (LAR) were
significantly higher in the C3 than C4 plants across all of
the N treatments, indicating the production of leaf area
using less biomass than in the C3 subspecies (Fig. 6b, c;
Table 1). When plants were grown under a decreased
supply of N, SLA was reduced in both subspecies, but
LAR declined in the C3 but not in the C4 subspecies.
These changes in LAR confirm our finding that the
allocation of growth to the leaves of C3 plants was
reduced at low N supply when plants were small, but
remained unaltered in the C4 subspecies.
Discussion
Partitioning of growth
Our data showed that C4 photosynthesis in A. semialata
was associated with a decrease in the allocation of
biomass to leaves compared with the C3 type (Figs 4b,
5c), supporting hypothesis ii; that greater efficiency at the
leaf scale requires a smaller canopy area. Despite this
smaller leaf canopy, higher rates of C4 than C3 photosynthesis at the same leaf N content (PNUE, Fig. 1) translated
into greater plant biomass production (Fig. 3a). However,
rather than increasing investment in roots compared with
the C3 type, reduced leaf and root growth in the C4 plants
allowed an increase in biomass partitioning to sexual
reproduction and corms (Fig. 4). Enlarged below-ground
corms in the C4 subspecies develop from the vegetative
culm and leaf bases, and probably function as storage
organs. These differences in growth partitioning between
photosynthetic types support the idea of a mechanistic
linkage between C4 photosynthesis and the ecological
strategies of plants. However, they suggest an additional
strategy to those proposed by Long (1999), indicating that
the high PNUE of C4 photosynthesis generated sufficient
carbon to allow greater investment in storage and sexual
reproduction. By contrast, the higher investment of
biomass in leaves and tillers in the C3 plants suggests the
predominance of vegetative growth and reproduction.
As nitrogen supply was reduced, above-ground biomass
decreased (Fig. 3c, d) in favour of root production
(Fig. 4a) in both subspecies. The response was, however,
no larger in the C3 than the C4 subspecies and refutes
hypothesis iii. A similar increase in root production has
been observed frequently in other C3 and C4 species
(Hocking and Meyer, 1991) and can be explained by plant
allometric response to treatment, rather than the direct
effects of treatment per se (Shipley and Meziane, 2002).
However, the consequences of this reallocation of growth
differed significantly between the C3 and C4 subspecies of
A. semialata, matching their contrasting growth patterns
Nitrogen-limitation in C3 and C4 plants 1711
Fig. 5. Allometric analyses of (a) leaf, (b) root, (c) leaf sheath, (d) corm, (e) average flower mass, and (f) numbers of flowers for C3 (closed symbols
and solid line) and C4 subspecies (open symbols and dashed line) of Alloteropsis semialata supplied 7 (squares), 3 (circles) or 0 (diamonds) g N m2
year1 nitrogen.
Table 3. Linear effects model for the apportioning of biomass to leaves, roots, corms, and culms in C3 and C4 subspecies of
Alloteropsis semialata
Analyses accounted for the interaction of subspecies with total plant size.
Parameter
Subspecies
Whole plant mass
Subspecies3whole plant
mass
Leaf biomass
Root biomass
Leaf sheath mass
Corm mass
Flower mass
Number of flowers
Average flower mass
n.s
n.s
n.s
n.s
n.s
n.s
F¼122.78, P <0.0001
F¼477.27, P <0.0001
F¼642.95, P <0.0001
F¼162.5, P <0.0001
F¼153.04, P <0.0001
F¼23.34, P <0.0001
F¼42.48, P <0.0001
F¼4.91, P <0.033
F¼63.32, P <0.0001
F¼13.92, P <0.0006
n.s.
F¼8.31, P <0.0001
F¼22.83, P <0.0001
F¼40.66, P <0.0001
n.s.
by reducing sexual reproduction in the C4, and leaf
production in the C3 type. Reduced N supply therefore
caused a decrease in canopy biomass and carbon
assimilation for the C3 subspecies that was avoided by
the C4 type.
Growth and development in this species have also been
monitored over two growing seasons in a common garden
experiment (Ripley et al., 2007). In each year, all 20 of
the C4 plants flowered over the November–December
period, while only one individual C3 plant flowered over
the whole experiment (Ibrahim et al., 2007). Since the
destructive harvest in our nitrogen-limitation experiment
was timed to coincide with this annual phase of sexual
reproduction, the strong contrast in biomass partitioning
between subspecies therefore suggests an ecologically
significant difference in allocation strategy, rather than an
1712 Ripley et al.
partitioning primarily through its effect on plant size. The
differences between subspecies result from differences in
allometric trajectories for many of the measured biomass
compartments. Similar size-mediated effects on biomass
partitioning have been demonstrated previously for nitrogen supply (Müller et al., 2000), atmospheric CO2
concentration (Coleman et al., 1993), branch crowding
(Weiner, 2004), and sowing density (Weiner, 1988).
However, other researchers have shown that allometric
relationships are directly altered by levels of nitrogen and
irradiance supply (Grechi et al., 2007). Further experimentation with sequential harvests in our model system
would be required to confirm the lack of direct treatment
effects on allometric relationships, and to quantify
ontogenetic drift.
Fig. 6. (a) Leaf area, (b) specific leaf area (SLA), and (c) leaf area ratio
(LAR) for C3 (closed symbols) and C4 (open symbols) subspecies of
Alloteropsis semialata supplied the indicated levels of N. All are mean
values with vertical bars representing standard errors (n >6).
ontogenetic response to nitrogen treatment (Coleman
et al., 1993). Furthermore, non-destructive measures of
leaf area measured at 4-week intervals during the course
of this experiment indicated that the relative growth rate
of leaf area was close to zero and not statistically different
between subspecies or treatments at the time of the plant
harvests (data not shown). This indicates that both
subspecies had reached the end of their vegetative growth
phase and supports the interpretation of differences being
due to allometry and not ontogeny.
Allometric analyses suggested that the primary effect of
nitrogen treatments was a shift along unique allometric
trajectories for each photosynthetic type, the magnitude
of which was not directly proportional to nitrogen supply (Fig. 5). Hence, nitrogen supply determined biomass
Productivity and nitrogen-use
The higher potential productivity of C4 than C3 plants is
well documented in the literature. Growth rates of C4
crops are ;1.5–2 times higher than C3 crops, whether
determined in the short-term at near-optimal conditions
(Ludlow, 1985) or over the entire growing season
(Monteith, 1978). Comparisons between closely related
species are less common, but Rajendrudu and Ramam Das
(1982) showed that the C4 Cleome gynandra had growth
rates approximately double those of the C3 C. viscosa,
despite a smaller leaf area in the C4 plants. Similarly, our
experiment showed that the C4 subspecies of A. semialata
produced more biomass at high N supply over a 23 week
period than the C3 subspecies (Fig. 3a), despite having
a smaller leaf area (Fig. 6a). The higher biomass production of the C4 subspecies was correlated with higher
photosynthetic rates rather than a lower cost of leaf
production (SLA) or a greater allocation of plant biomass
to leaves (Fig. 6b, c; Table 1). The C4 mechanism enabled
the plants to accumulate 1.4 times more carbon per unit
leaf area than the C3 plants for the same investment in leaf
nitrogen (Fig. 1). In addition the C4 plants produced less
leaf area (Fig. 6), reducing leaf nitrogen and carbon costs.
These savings allowed the C4 subspecies to partition
a greater proportion of biomass to storage and sexual
reproduction, and allowed for the production of leaves
with a lower SLA.
N limitation occurred at the scale of the plant rather
than the leaf in A. semialata subspecies. As N supply was
reduced, area-based leaf N content, CO2 assimilation rate,
and PNUE remained unchanged in both the C3 and C4
subspecies, despite large decreases in whole plant biomass
(Figs 1a, b, c, 3a). These data indicate a scaling of growth
to match the N supply rather than a limitation of
physiological processes by reduced leaf N. A similar
response has been noted for species such as potato (Vos
and Biemond, 1992) and contrasts with the response of
maize, where nutrient availability causes stronger responses
Nitrogen-limitation in C3 and C4 plants 1713
in PNUE and leaf photosynthesis than leaf area (Vos
et al., 2005).
Ecological implications
We suggest that both subspecies of A. semialata are
adapted to low soil nutrient conditions, and hypothesize
that their contrasting growth strategies may be adaptive in
relation to differing disturbance frequencies. The C4
subspecies occurs typically in mixed C3–C4 grassland
communities under high rainfall regimes in South Africa
(Ellis et al., 1980). These ecosystems experience frequent
fire because a high biomass fuel load accumulates during
the growing season, then dries during the winter (Bond,
1997). Frequent fire depletes ecosystem N stocks through
the volatilization of organic matter, and high rainfall
causes leaching, making N a limiting resource for plant
growth in mesic grasslands (Blair et al., 1998). High
assimilation rates and fast growth may be advantageous in
a post-fire environment in the absence of competition for
light, with the below-ground storage of resources and
prolific sexual reproduction facilitating resprouting and
recruitment. Competition after fires is a crucial determinant of community structure in the fire-prone mesic
grasslands of North America, and C4 grasses dominate
these ecosystems by achieving rapid regrowth with limited
N resources (Collins and Steinauer, 1998).
The C3 subspecies of A. semialata co-occurs with the
C4 type in the southern mesic grasslands of South Africa,
but its range extends to higher latitudes and elevations
characterized by lower temperature and rainfall (Ellis
et al., 1980). These regions are typically drier, and subject
to less frequent burning because the primary production of
a fine fuel load by grasses is lower (Bond, 1997), and
related to infertile soils of quartzite and andesitic origin.
We suggest that an ecological strategy favouring vegetative reproduction and leaf growth may be advantageous in
the closed canopies of these ecosystems by facilitating the
occupation of space and competition for light. Further
experimentation is required to determine the relationship
between photosynthetic pathway, growth partitioning
strategies, and the response to fire in A. semialata
subspecies.
Conclusions
A unique model system has been used here to demonstrate
a link between the differing photosynthetic N-use efficiencies and carbon assimilation in C3 and C4 grasses and
contrasting strategies of growth. Our data show for the
first time that the greater efficiency of C4 photosynthesis
may be associated with higher productivity and increased
investment in sexual reproduction and below-ground
storage than the C3 type, despite reduced growth allocation to the leaf canopy and roots. These findings highlight
important potential advantages of the pathway in infertile
ecosystems disturbed frequently by fire.
Acknowledgements
We gratefully acknowledge funding from the following sources: the
South African National Research Foundation (NRF) and the Rhodes
University JRC (BSR and TA); and a Royal Society University
Research Fellowship (CPO). We are grateful to Andrew Beckerman
for his advice on statistical analyses.
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