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2Departamento de Biodiversidad y Biología Experimental, F.C.E. N., Universidad de Buenos Aires, Ciudad Universitaria, 1428 Capital Federal, Argentina; 3Department of Crop Science, P.O. Box 7620, North Carolina State University, Raleigh, North Carolina 27695-7620 USA; 4Division of Environmental Science and Policy, Nicholas School of the Environment & Earth Science, and Department of Biology, Phytotron Building, Box 90340, Duke University, Durham, North Carolina 27708-0340 USA
Received for publication July 11, 2005. Accepted for publication February 23, 2006.
ABSTRACT
Predicting future plant and ecosystem responses to elevated CO2 also requires an understanding of the role of other factors, especially soil nitrogen. This is particularly challenging for global aridlands where total N and the relative amounts of nitrate and ammonia vary both spatially and seasonally. We measured gas exchange and primary and secondary C metabolites in seedlings of two dominant aridland shrub species (Prosopis flexuosa [S America] and P. glandulosa [N America]) grown at ambient (350 ppm) or elevated (650 ppm) CO2 and nitrogen at two levels (low [0.8 mM] and high [8.0 mM]) and at either 1 : 1 or 3 : 1 nitrate to ammonia. Whereas elevated CO2 increased assimilation rate, water use efficiency, and primary carbon metabolites in both species, these increases were strongly contingent upon nitrogen availability. Elevated CO2 did not increase secondary metabolites (i.e., phenolics). For these important aridland species, the effects of elevated CO2 are strongly influenced by nitrogen availability and to a lesser extent by the relative amounts of nitrate and ammonia supplied, which underscores the importance of both the amount and chemical composition of soil nitrogen in mediating the potential responses of seedling growth and establishment of aridland plants under future CO2-enriched atmospheres.
Key Words: ammonia-N • global change • nitrate-N • phenolic compounds • Prosopis flexuosa • Prosopis glandulosa • rangeland
Future increases in the CO2 concentration of the atmosphere are expected to have a marked influence on plant physiology and ecosystem functioning (e.g., Drake et al., 1997
; Ward and Strain, 1999
). However, there is strong evidence that many species of plants have already responded to the 30% increase in atmospheric CO2 concentration that has occurred during the past 200 yr (Ward and Strain, 1999
), including changes in physiology (Long et al., 2004
), growth (Ainsworth and Long, 2005
), chemical composition of plant tissues (Peñuelas and Estiarte, 1998
), and phenology (Navas et al., 1997
). A major challenge in predicting plant responses to elevated CO2 is understanding the complex relationships between these effects and other factors, especially nutrient availability, which plays a central role (Long et al., 2004
). For example, numerous observations show that growth responses to elevated CO2 are lessened or even eliminated under low N nutrition (e.g., Cure et al., 1989
; Jackson and Reynolds, 1996
; Drake et al., 1997; Stitt and Krapp, 1999
; Tognetti and Johnson, 1999
; Paul and Foyer, 2001
; Ainsworth and Long, 2005
; Hu et al., 2005
). This effect is the result of the coordination that exists between C and N regulatory systems in the plant (Rufty, 1997
). This coordination may involve signals derived from both C and N metabolism that interact to regulate N uptake and assimilation, organic acid synthesis, starch accumulation, photosynthesis, and biomass allocation under different CO2 x N regimes (Stitt and Krapp, 1999
; Stitt et al., 2002
). While the signals appear linked with the N status of the plant, their modulation may also be influenced by the relative amounts of nitrate (NO3–) and ammonia (NH4+) supplied (Cruz et al., 1997
; Geiger et al., 1999
; Gloser et al., 2002
; Plhak, 2003
).
Much of our present understanding about plant responses to the interacting effects of elevated CO2 and N nutrition comes from studies with crops or species from mesic ecosystems (Körner, 1996
). Much less information is available regarding responses of plants from natural ecosystems, especially for arid and semiarid regions. Aridlands, which cover over 40% of the global land surface (Bailey, 1998
), are characterized by large spatial and seasonal variations in total N availability (Martin and Asner, 2005
), as well as the relative amounts of NO3– to NH4+ as the main sources of nitrogen (hereafter denoted as ) (Virginia et al.; Mazzarino et al., 1991
b; Bennett and Adams, 1999
).
This paucity of data in arid and semiarid regions motivated us (Causin et al., 2004
) to study the influence of N nutrition on plant responses to elevated CO2 concentrations, specifically to see if variations in and total N available (Nlevel) would alter various growth and physiological responses of seedlings of Prosopis glandulosa Torr. and P. flexuosa DC (Fabaceae). These two species dominate many aridland communities of North and South America, respectively. Along with several closely related legumes, these species are major components of woody shrub encroachment, which in the past 100 yr has affected many regions of the globe (for P. flexuosa, see Pisani and Distel, 1998
; for P. glandulosa, see Van Auken and Bush, 1997
). In these arid rangeland ecosystems, Prosopis seedlings are heavily browsed by cattle and other herbivores throughout the year (e.g., Guevara et al., 1996
; Van den Bosch et al., 1997
; Martínez and López-Portillo, 2003
). Our previous study (Causin et al., 2004
) focused on various aspects of N metabolism, growth measures, N uptake, N metabolites (e.g., free NO3–, soluble proteins), and organic acids (which is an indicator of the ability of species to use nitrate vs. ammonia as their primary source of N, e.g., Salsac et al., 1987
). We found that both species were highly responsive to Nlevel and that under conditions of limiting N supply, the species benefited from increases in CO2, provided that a high proportion of NO3–-N to NH4+-N is present in the soil solution.
Further research on the C and N metabolism of this genus is needed to better distinguish between the direct effects of elevated CO2 and N limitations and their interactions. For example, while Polley et al. (1996)
showed that, under drought conditions, the survival of P. glandulosa seedlings was increased by 40% by increasing the CO2 concentration, how would variability in soil Nlevel and affect these survival rates? In this paper we extend the work of Causin et al. (2004)
, which focused on growth and N metabolism, by addressing questions related to the C metabolism of P. glandulosa and P. flexuosa. Our primary motivation is to explore how elevated CO2 and nitrogen availability potentially interact to alter various aspects of the carbon metabolism of these species. As in our previous study, seedlings were grown at two concentrations of CO2 (350 and 650 ppm), two values of Nlevel (low, 0.8 mM, and high, 8.0 mM), which were supplied at either 1 : 1 or 3 : 1 NO3– : NH4+ ratios (). Using this experimental design, we examine impacts and interactions on the following: (1) Net photosynthesis and photosynthetic water use efficiency (PWUE); (2) Production of primary carbon metabolites (i.e., soluble sugars, starch, total nonstructural carbohydrates); and (3) Production of secondary carbon metabolites (i.e., total phenolics and their protein-precipitating capacity, which serve as a measure of plant antiherbivore defense).
With regard to secondary metabolites, elevated CO2 is known to increase phenolics (Lincoln et al., 1993
; Lindroth, 1996
). While the concentration of leaf phenolic compounds is affected by numerous other factors, including light intensity and quality, nitrogen availability, herbivory, and oxidative stress (Dudt and Shure, 1994; Larson, 1995; McKee, 1995; Kinney et al., 1997
; Tognetti and Johnson, 1999
a; 1999c), to our knowledge, there is almost no information on how changes in —in conjunction with increasing levels of atmospheric CO2 concentrations—may affect the production of phenolic compounds. Our experimental design enables us to specifically examine how changing carbon and nitrogen availabilities affect leaf phenolic concentration under highly controlled conditions.
MATERIALS AND METHODS
Experimental design
The present study is focused on the seedling phase (up to ca. 1 year in Prosopis, as per Brown and Archer, 1990
) for the following reasons, which are specifically related to the two species of Prosopis used: (1) the seedling phase is most crucial for the successful establishment and growth of Prosopis in grass-dominated communities (Brown and Archer, 1989
; Brown and Archer, 1990
); (2) seedling growth is markedly dependent upon mineral N nutrition (e.g., Causin et al., 2004
; Hahne and Schuch, 2004
) (Note that we did not use plants infected with Rhizobium; evidence suggests that symbiotic N2-fixation in these species does not occur until later stages of development. [Jenkins et al., 1988
]); and (3) it is during the first month of growth and development, following germination, when elevated CO2 has some of its largest impacts (Polley et al., 1996
; Causin et al., 2004
; Derner et al., 2005
).
A detailed description of plant growth conditions is given in Causin et al. (2004). Briefly, half-sib seed families of P. glandulosa and P. flexuosa were sown in PVC pots containing a mixture of sand to gravel (4 : 1). At 8 d after sowing, 12 pots per species were transferred to each of four walk-in growth chambers to create a 2 spp. x 2 CO2 levels x 2 Nlevel x 2. Two chambers were maintained at 350 ppm CO2 (ambient controls) and two at 650 ppm (elevated) CO2. Within each chamber, four Nlevel x treatments were applied to three pots per species (1 plant/pot). CO2 control in these chambers was maintained using a centralized CO2 control system with specially designed hardware and software and LiCor (Lincoln, Nebraska, USA) gas analyzers. Chamber CO2 levels are sampled every 3–7 min, and injection rates of pure CO2 are controlled to maintain CO2 setpoints in the chambers. In general, this system is able to maintain CO2 levels within 5% of the setpoint. Pots were irrigated three times per week with a modified Hogland's nutrient solution (see Causin et al., 2004
) with the specified N concentrations. Pots were flushed every 2 weeks with distilled water to minimize any nutrient or salt accumulation.
Plant harvests
Plants (six replicates per treatment combination) were harvested at 56 days after sowing. A subsample of the leaves was frozen at –70°C for later freeze drying, and the remaining tissue dried at 65°C to a constant mass. Stems were separated from roots and dried at 65°C. Roots were removed from the pots and carefully rinsed to remove soil. The upper, thickest portion of the root was separated and dried at 65°C; the remaining root tissue was kept at –70°C and freeze-dried. After recording all dry masses, the freeze-dried leaf and root tissues were finely ground in a Tecator cyclonic mill (Cyclotec, Herndon, VA).
Gas exchange
Four to 7 days prior to harvesting, leaf gas exchange (stomatal conductance, photosynthesis, and transpiration) of each plant was measured using a Li-Cor 6400 portable photosynthesis system. Two mature leaves (1/day) on each plant were measured on two different days between 0900 and 1400 hours (i.e., when maximum photosynthetic rate was expected under our experimental conditions). Measurements were performed at 900 µmol · m–2 · s–1 light intensity and 40% relative humidity. Data from light-saturated net photosynthetic rate (Asat; µmol CO2 · m–2 · s–1) and transpiration rate (E; mmol H2O · m–2 · s–1) were used to estimate photosynthetic water use efficiency (PWUE = Asat/E). The mean of the two measurements per plant was used for the statistical analysis.
Chemical analyses
Primary carbon metabolites
Freeze–dried leaf and root material was used for the analysis of tissue metabolites. Because single plants cannot provide adequate tissue volume, samples of roots and leaves from two randomly chosen plants from the same treatment (one plant from each of the two CO2 level chambers) were pooled to create three samples of sufficient size. All chemical analysis were performed on these three "compound replicates" unless otherwise stated.
For carbohydrate analyses, 25 mg samples were extracted three times with 8.0 mL 80% ethanol solution. The resulting pellet was frozen at –18°C for starch analysis (described later), and the combined supernatants were made up to 25 mL with the ethanol solution. Aliquots of 5.0 mL were dried overnight in a speedvac (Savant Speedvac Concentrator, Thermo-Savant Co., Holbrook, New York, USA) and the residue was reconstituted in HPLC-grade distilled water for the determination of soluble sugars using a Dionex Ion Chromatography system (Dionex, Sunnyvale, California, USA). A 10-µL sample of each extract was injected into a stream consisting of a gradient of 200 mmol/L NaOH and HPLC-grade water. The saccharides (glucose, fructose, and sucrose) were separated by HPAEC (high-performance anion-exchange chromatography) after passing through a CarboPac PA10 (Dionex Corp., Sunnyvale, CA) guard and analytical column (4 x 250 mm), and detected via pulsed amerometric detection. Peak areas for each component were quantified by PeakNet ver. 5.21 chromatography software (Dionex) and compared to reference standards.
Starch content was determined as glucose equivalents (Tissue and Wright, 1995
) after hydrolysis with 8.0 mL 35% perchloric acid.
Secondary carbon metabolites
Phenolic compounds in the leaves were determined following the procedure described in Pisani and Distel (1998)
. Phenol concentrations were expressed as absorbance per unit dry mass, as recommended by Martin and Martin (1982)
.
The capability of certain phenolic compounds to precipitate proteins is thought to be responsible for the adverse effects which these substances have for many consumer organisms. Hence a bovine serum albumin (BSA) precipitating test on phenolic extracts from leaves was conducted as a general measure of a plant's investment in, or efficacy of, antiherbivore defense (Martin and Martin, 1982
). Briefly, 0.7 mL samples of the phenolic extracts were vortexed with 0.8 ml BSA solution (1 mg/mL in acetate buffer 0.2 mol/L, pH 5.0, containing 0.17 mol/L NaCl) and allowed to stand at room temperature for 15 min. After centrifugation (19000 x g, 10 min, 5°C), the pellet was rinsed with 0.4 mL acetate buffer, centrifuged as before, and dissolved in 2.0 mL 1% SDS in 5% triethanolamine. After the solution stood for 15 min, precipitated phenols were determined by adding 0.5 mL 0.1 mol/L FeCl3 in 0.1 N HCl and reading absorbance at 610 nm against a distilled water blank.
Statistical analyses
All analyses were performed using Data Desk software (Velleman, 1995
). Effects of treatments were analyzed using a nested analysis of variance (ANOVA) model (Potvin and Tardif, 1988
). The model included CO2, species, Nlevel, and as main effects, with chamber number as a random factor nested within CO2. Each pot (i.e., an individual) is considered an experimental unit and for the analysis of pooled samples, each "compound replicate" is the experimental unit. In the nested design, the chamber is the experimental unit for CO2 comparisons. A four-way ANOVA design (using the same main effect terms) was used.
Histograms and normal probability plots were used to determine whether data required logarithmic, square-root, or arcsine square-root transformation for satisfactorily normal distributions of residuals and homogeneity of variances. Post-ANOVA comparisons of treatment means were made using Bonferroni's test (Day and Quinn, 1989
) All P values given in the text refer to results from the ANOVAs, unless otherwise indicated. Differences were considered significant at P < 0.05.
RESULTS
Gas exchange
The light-saturated, maximum net photosynthetic rate (Asat) was markedly affected by total N supply (Nlevel main effect, P < 0.001), though the effect depended on the atmospheric CO2 concentration as indicated by a significant CO2 x Nlevel interaction term (P < 0.001). Posthoc tests showed that, at high N supply, Asat was significantly higher at elevated CO2 as compared to the ambient control, while at low N supply the opposite was true (Fig. 1a, b). There was a significant difference in Asat between the species (P < 0.01), primarily reflecting somewhat higher photosynthetic rates in P. glandulosa in some of the treatments. No significant effects on Asat were found.
|
The combination of the described treatment effects on assimilation and conductance resulted in increased PWUE at elevated CO2 for most of the treatments, with one exception: low N at a 1 : 1 (Fig. 1e, f). The PWUE also generally increased at high Nlevel irrespective of other treatment factors (P < 0.001 for Nlevel main effect), so that the highest PWUE values were found in plants grown at high Nlevel and elevated CO2 (Fig. 1e, f). The high NO3– : NH4+ ratio also significantly increased PWUE as compared to the 1 : 1 ratio (P < 0.01), and this effect was most apparent at elevated CO2 (P < 0.05 for CO2 x interaction term). PWUE was higher in P. glandulosa than in P. flexuosa (P < 0.01), principally because of lower stomatal conductances.
Primary carbon metabolites
The starch content in plant tissues was markedly affected by the different treatments, and we also found differences in treatment effects on leaves vs. roots. Elevated CO2 had a very strong effect on starch content of leaves, resulting in 20–88% more starch accumulation as compared to ambient CO2 levels (Fig. 2a, b). However, there was no significant CO2 effect on starch level in the roots (Fig. 2c, d). There were also highly significant (P < 0.001) effects of Nlevel on starch content of both leaves and roots, with generally lower starch contents at high Nlevel (Fig. 2a–d). Significant (P < 0.01) three-way interactions among Nlevel, , and species (leaf starch), and among Nlevel, , and CO2 (root starch) indicate that both N supply and the source ratio affects starch contents, but the specific effect depends upon species and/or CO2 level.
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Treatment responses of leaf total nonstructural carbohydrates (TNC) paralleled those of starch, while in the roots the pool of TNC significantly increased with elevated CO2 (data not shown). At low Nlevel, P. glandulosa had significantly higher TNC concentration in the roots than P. flexuosa plants (P < 0.05 for the species x Nlevel interaction).
Secondary carbon metabolites
In general, the concentration of phenolic compounds in the leaves was significantly affected by most of the treatment factors. However, the patterns of these effects were complicated by numerous two- and three-way interactions among treatments. Our primary finding is that, for most cases, phenolics concentration consistently increased at high Nlevel and 1 : 1 (Fig. 3a, b). The only exception was for P. flexuosa plants when grown at ambient CO2; the highest leaf phenolics concentration was also found at high Nlevel, but at the 3 : 1 (Fig. 3a). No significant correlations were found between the concentration of phenolic compounds and the concentration of soluble sugars or TNC in the leaves (data not shown).
|
Apart from structural phenolics such as lignin, soluble phenolic compounds are amongst the most abundant C-based secondary metabolites in the leaves of many plant species (Peñuelas and Estiarte, 1998
, and references therein). Because their synthesis depends upon both C and N status (Jones and Harley, 1999
), we plotted total phenolic concentrations, as well as the concentration of the phenolic compounds with BSA-precipitating capacity, vs. the ratio of total N concentration (data from Causin et al., 2004
) to TNC (i.e., N/TNC). There was a weak positive relationship between total phenolics and N/TNC for plants grown with a 1 : 1 , whereas for plants grown at the 3 : 1 ratio, phenolics remained unchanged as N / TNC increased (data not shown). In contrast, the BSA-precipitating capacity of the extracted phenols consistently increased with increasing N/TNC ratio in all the conditions tested (Fig. 4).
|
Soil nitrogen availability is one of the key factors controlling seedling establishment in semiarid ecosystems (Whitford et al, 2001
), and both Prosopis species are highly responsive to N supply (Causin et al., 2004
). As previously shown for whole plant growth, the effect of elevated CO2 on net C assimilation rate is also strongly mediated by N availability. Elevated CO2 increased net assimilation rate at high Nlevel, but decreased it at a low N supply. These results are consistent with many other studies showing the effect of nutrient availability on plant responses to elevated CO2 (e.g., Geiger et al., 1999
; Stitt and Krapp, 1999
).
Drake et al. (1997)
proposed that the primary response of plants to rising atmospheric CO2 is an increase in resource use efficiency. Indeed, we found that elevated CO2 affected photosynthetic water use efficiency (PWUE) in both species of Prosopis, although this effect was also modified by N availability. Moreover, PWUE was increased by elevated CO2 particularly under conditions of high NO3–N (see Fig. 1e, f). This observation agrees with other studies that report a positive correlation between PWUE and NO3– availability, particularly under limiting or moderate water supply (Raven et al., 1992
; Ashraf and Habib-ur-Rehman, 1999). Although water availability was not limited in our experimental conditions, a high NO3-N supply consistently led to a decrease of stomatal conductance (refer to Fig. 2c, d) and favored the accumulation of free NO3– and organic acids in the leaves (Causin et al., 2004
) when plants were grown at elevated CO2. Hence, it is possible that the observed increment in PWUE involved a reduction of transpiration associated with reduced leaf water potentials (e.g., McIntyre, 1997; Cárdenas-Navarro et al., 1999). This response may in part account for the beneficial effects of a high NO3-N supply mentioned previously, as well as contribute to the success of seedling establishment in their natural environment.
We found an increase in leaf starch and an increase in the ratio of leaf starch to sucrose at elevated CO2 levels for both species (Fig. 2a, b). These increases are frequently associated with feedback inhibition of photosynthesis and/or growth (e.g., Wulff and Strain, 1982
; Grimmer et al., 1999
; Paul and Foyer, 2001
). However, the effects of increased starch accumulation on carbon acquisition were strongly dependent upon plant N status—that is, at a high Nlevel, both photosynthesis and plant growth were stimulated by elevated CO2 (Causin et al., 2004
) despite the increase in leaf starch to sucrose ratio levels. These results are consistent with other studies (e.g., Rogers et al., 1996a
, b; Geiger et al., 1999
; Ward and Strain, 1999
) and support the hypothesis that, with adequate N supply, there is no major decrease of the internal concentration of N and specific N metabolites that might be involved in the modulation of the sugar-mediated repression of photosynthesis (Geiger et al., 1999
; Stitt and Krapp, 1999
). The positive effect of N nutrition on carbon acquisition at elevated CO2 might be enhanced by the presence of a high nitrate supply because NO3– ions have been shown to inhibit starch synthesis and accumulation (Scheible et al., 1997
; Stitt et al., 2002
). Even though NO3-N stimulated seedling growth at elevated atmospheric CO2 when N supply was limiting, we did not found a consistent relationship between NO3– availability and starch accumulation, at either N levels.
In addition to affecting primary metabolism, changes in N and C availabilities may also influence the production of secondary metabolites such as phenolics, alkaloids, and other compounds often regarded as related to antiherbivore defense (Julkunen-Tiitto et al., 1993
; Kinney et al., 1997
; Gebauer et al., 1998
; Peñuelas and Estiarte, 1998
). Under low nutrient (particularly N) availability, growth potential may be more affected than photosynthetic potential and, in such circumstances, any increase in C would be diverted to the accumulation of secondary (defense) metabolites (i.e., the carbon/nutrient balance hypothesis, see Bryant et al., 1983
; Chapin et al., 1987
). However, we found that the extra C gain due to elevated CO2 stimulated primary metabolic responses (e.g., increased carbohydrates) rather than phenolic accumulation. Moreover, in P. glandulosa plants elevated CO2 significantly increased carbohydrates (TNC) allocation to the root system when N supply was limiting. Even though an increased TNC allocation to the root system may contribute to enhance nutrient uptake capacity (e.g., BassiriRad, et al., 1996; BassiriRad et al., 1997) this effect was not accompanied by a consistent increase in root growth and/or N acquisition, indicating that other factors were limiting this process under our experimental conditions (see Causin et al., 2004
). Overall, the present results are not particularly supportive of the carbon C–nutrient balance hypothesis (Bryant et al., 1983
), which suggests that in situations with limiting nutrients and abundant light, plants will shunt "excess" C into C-based defenses such as phenolics (Hamilton et al., 2001
; Koricheva, 2002
). In a study where water and nutrient availabilities were manipulated, Pisani and Distel (1998)
also found that phenolic production in P. flexulosa plants could not be explained according to the C–nutrient balance hypothesis.
As noted, elevated CO2 did not increase phenolic concentrations in the two Prosopis species studied; rather, soil N availability (Nlevel) and source () had the greatest effects on phenolics. The only treatment factor that strongly stimulated phenolic accumulation was high Nlevel, and mostly at 1 : 1 (Fig. 3a, b). Furthermore, significant positive correlations did not exist between the concentrations of total phenolics and soluble sugar or TNC accumulation (data not shown), as reported for other species (Gebauer et al., 1998
; Peñuelas and Estiarte, 1998
). While concentration data per se do not allow us to discriminate whether a higher accumulation of phenolic compounds per unit mass is actually due to an increased synthesis (i.e., higher production) or decreased turnover of tissue phenolics, the net result is an increased C allocation to this secondary metabolite pool. Therefore, assuming that phenolics may act as chemical defense compounds, an increment in N rather than C availability would be more efficient in raising the amount of chemical defenses per unit mass (be it for growth or consumed) in the studied species.
Traditionally, leaf concentration of phenolics is considered to be a function of either TNC (Peñuelas and Estiarte, 1998
) or nitrogen concentration (or soluble proteins, as per Jones and Harley, 1999
). Because of this intrinsic interdependency, we examined the relationships between phenolics and the relative proportion of N to TNC in the leaves. Many studies have shown that with elevated CO2, the concentration of leaf N decreases while TNC, and C-based secondary metabolites increase (see Drake et al., 1997
; Peñuelas and Estiarte, 1998
). Even though we did not find a consistent relationship between soluble phenolics concentration and N/TNC ratio in our experimental conditions, we did find that the BSA-precipitating phenolic fraction increased significantly with N/TNC ratio (see Fig. 4). Martin and Martin (1982)
showed that the protein-precipitating capacity of the phenolic extracts provides a more reliable measure of their utility as defensive chemicals rather than total phenolic content. There is also some evidence indicating that different herbivore species forage preferentially on leaves with high N to C ratios (e.g., Ball et al., 2000
and references therein). Hence, in cases where leaf N to C ratio is high (i.e., high soil N), it is feasible that for the Prosopis species studied, leaves would also have a higher proportion of protein-binding phenolics, which could hinder herbivory.
In conclusion, for both Prosopis glandulosa and P. flexuosa, the effects of elevated CO2 are strongly mediated by N availability (Nlevel) and, to a lesser extent by the relative amounts of nitrate and ammonia in the N supplied (). In fact, for every physiological and biochemical response variable examined, Nlevel alone, or Nlevel interacting with CO2 concentration and/or , proved to have a much greater effect than elevated CO2 alone. Studies of P. glandulosa in the Sonoran Desert indicate that these shrubs are able to obtain 43–61% of their total plant N via symbiotic N fixation (Shearer et al., 1983
), suggesting a low reliance on soil N for most of its long adult life. Nevertheless, the results of this study, when combined with our previous work (Causin et al. 2004
), underscore the importance of both the amount and chemical makeup of soil N to seedling establishment.
FOOTNOTES
1 The authors thank D. Ravetta and A. Vilela (Faculty of Agronomy, University of Buenos Aires) for providing seeds of Prosopis flexuosa, P. Kemp (University of San Diego) for helpful comments on the manuscript, and D. Tremmel (Duke University) for the statistical analyses. This research is supported in part by the René H. Thalmann Program of the University of Buenos Aires (Expte. #102.768/58 Anexo 658), USDA Specific Cooperative Agreement 58-1270-3-070, and grant IBN-9985877 from the National Science Foundation in support of the Phytotron at Duke University. 
5 Author for correspondence (james.f.reynolds{at}duke.edu
)
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]). Lines represent linear regressions of y on x for each ratio (1 : 1 = y1 and solid lines; 3 : 1 = y2 and dashed lines).