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 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
