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. 2009 Nov;151(3):1667–1676. doi: 10.1104/pp.109.145870

Plant δ15N Correlates with the Transpiration Efficiency of Nitrogen Acquisition in Tropical Trees1,[OA]

Lucas A Cernusak 1,2,*, Klaus Winter 1, Benjamin L Turner 1
PMCID: PMC2773072  PMID: 19726571

Abstract

Based upon considerations of a theoretical model of 15N/14N fractionation during steady-state nitrate uptake from soil, we hypothesized that, for plants grown in a common soil environment, whole-plant δ15N (δP) should vary as a function of the transpiration efficiency of nitrogen acquisition (FN/v) and the difference between δP and root δ15N (δP − δR). We tested these hypotheses with measurements of several tropical tree and liana species. Consistent with theoretical expectations, both FN/v and δP − δR were significant sources of variation in δP, and the relationship between δP and FN/v differed between non-N2-fixing and N2-fixing species. We interpret the correlation between δP and FN/v as resulting from variation in mineral nitrogen efflux-to-influx ratios across plasma membranes of root cells. These results provide a simple explanation of variation in δ15N of terrestrial plants and have implications for understanding nitrogen cycling in ecosystems.

Variation in the natural abundance of stable nitrogen isotopes (δ15N) in terrestrial plants can provide valuable information about plant nitrogen acquisition and ecosystem nitrogen cycling (Handley and Raven, 1992; Högberg, 1997; Evans, 2001; Robinson, 2001). However, interpretation of foliar or whole-plant δ15N data is complicated by the existence of multiple sources of nitrogen in the soil and the possibility for variable discrimination against 15N during the assimilation of each source (Handley and Raven, 1992; Evans, 2001). Analytical equations describing discrimination against 15N during uptake of nitrogenous solutes from the soil could greatly enhance the application of plant δ15N data in physiological and ecological investigations.

Comstock (2001) presented a theoretical model of 15N/14N fractionation during steady-state uptake of nitrate, a primary source of nitrogen for terrestrial plants. Here, we extend this model to formulate the hypothesis that plant δ15N should vary as a function of the transpiration efficiency of nitrogen acquisition for plants growing under similar soil conditions, and we test this with measurements on several tropical tree and liana species. Figure 1 shows a simplified model of nitrate uptake in plants based on that presented previously (Comstock, 2001). In the steady state, the following relationship can be defined:

graphic file with name M1.gif (1)

where FIn is the influx of nitrate from the soil into root cells, FEn is the efflux of nitrate from root cells back to the soil, FRn is the assimilation of nitrate into organic molecules in root cells, the first step of which is reduction of nitrate to nitrite by nitrate reductase, and FXn is the flux of nitrate from root cells into the xylem sap, which results in the transport of nitrate from the root to the shoot in the transpiration stream. Because nitrate is essentially absent from phloem sap exported from leaves in higher plants (Peoples and Gifford, 1997), the flux of nitrate into xylem sap in the roots is equal to the assimilation rate of nitrate in the shoot in the steady state. Assuming nitrate as the sole nitrogen source and that there is no nitrogen isotope fractionation associated with FIn, FEn, or FXn, discrimination against 15N during nitrogen uptake (ΔP) can be expressed as (Comstock, 2001):

graphic file with name M2.gif (2)

where FNn is the net uptake of nitrate from the soil by the plant, defined as FIn − FEn, or equivalently as FRn + FXn, and b is the discrimination constant for reduction of nitrate to nitrite by nitrate reductase, estimated to be about 15‰ (Ledgard et al., 1985; Handley and Raven, 1992; Tcherkez and Farquhar, 2006). The assumption of no isotopic discrimination associated with FIn, FEn, or FXn means that transport of nitrate across the plasmalemma membranes of root cells and transport of nitrate and organic nitrogen into the xylem sap are assumed to be nondiscriminating processes. A full derivation of Equation 2 and justification for the assumptions involved is given by Comstock (2001). The ΔP is defined as:

graphic file with name M3.gif (3)

where RSn is 15N/14N of soil nitrate, RP is 15N/14N of plant nitrogen, δSn is δ15N of soil nitrate, and δP is δ15N of plant nitrogen. Combining Equations 2 and 3 gives:

graphic file with name M4.gif (4)

Figure 1.

Figure 1.

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A simplified model of steady-state nitrate uptake, based on the conceptual model of Comstock (2001). FIn is the influx of nitrate from the soil solution into root cells; FEn is the efflux of nitrate from root cells back to the soil solution; FRn is the assimilation flux of nitrate into organic molecules within root cells; and FXn is the flux of nitrate from root cells into the xylem. Nitrate loaded into the xylem is carried to the leaf in the transpiration stream, where it can also be assimilated into organic molecules. Because nitrate is not exported from leaves in the phloem, the flux of nitrate into the xylem in the root is equal to the assimilation flux in the shoot in the steady state. The discrimination against 15N during nitrate reduction in both the root and the shoot is defined as b, which is a constant. FIn, FEn, and FXn are assumed to proceed without discrimination against 15N.

Equation 2 results from the combination of individual discrimination expressions for root-assimilated nitrate and leaf-assimilated nitrate (Comstock, 2001). The expression for discrimination against 15N during assimilation of nitrate in the root (ΔRn) is given by ΔRn = (1 − FRn/FIn)b. That for discrimination against 15N during assimilation in the leaf (ΔLn) is given by ΔLn = −(FRn/FIn)b. Subtracting ΔLn from ΔRn yields the expression ΔRn = ΔLn + b. Finally, applying the approximations that ΔRn ≈ δSn − δRn and ΔLn ≈ δSn − δLn leads to the following simple expression: δRn = δLn − b, where δRn is δ15N of root-assimilated, nitrate-derived nitrogen and δLn is δ15N of leaf-assimilated, nitrate-derived nitrogen.

The relationship between δRn and δLn can be intuitively understood by considering two contrasting examples. As a first example, consider the situation when FRn is small compared with FIn. In this case, the cytosolic nitrate pool in the root cells will be little enriched compared with nitrate in the soil solution, while at the same time the discrimination by nitrate reductase in the roots will be nearly fully expressed, because the supply of nitrate is large compared with consumption by the discriminating enzyme. In this case, root-assimilated nitrogen will show a large discrimination relative to soil nitrate, whereas nitrate loaded into the xylem, destined for assimilation in the shoot, will show a small enrichment relative to soil nitrate. In the steady state, assimilation of nitrate in the shoot proceeds without discrimination, because there is no branch point in the reaction sequence. That is to say, there is only one possible fate for nitrate that has been loaded into the xylem under steady-state conditions.

As a second example, consider the situation when FRn approaches the value of FIn. Here, the cytosolic nitrate pool will show a large enrichment relative to soil nitrate that approaches b, while at the same time discrimination by nitrate reductase in the root will be very small, because nearly all of the available substrate is being consumed. In this case, root-assimilated nitrogen will have a δ15N only slightly depleted compared with soil nitrate, whereas nitrate loaded into the xylem will show a large enrichment that approaches b relative to soil nitrate. In both examples, the difference between δRn and δLn will be approximated by the value of b.

We further suggest that for plants grown on nitrate, the nitrogen pool in the plant (NP) can be partitioned into a root-assimilated pool (NRn) and a shoot-assimilated pool (NLn).

graphic file with name M5.gif (5)

Equation 5 assumes that the pool of unassimilated nitrate in the plant, stored in vacuoles, for example, is negligible. Storage of nitrate typically accounts for less than 1% of total plant nitrogen in uncultivated plants (Smirnoff and Stewart, 1985; Pate et al., 1993; Schmidt and Stewart, 1997; Aidar et al., 2003); thus, this assumption should generally be valid. However, exceptions can occur, for example, in crop plants grown in hydroponics (Evans et al., 1996; Seginer, 2003). Equation 5 can be written for 15N as:

graphic file with name M6.gif (6)

where RP is 15N/14N of the plant nitrogen pool, RLn is 15N/14N of the shoot-assimilated pool, and RRn is 15N/14N of the root-assimilated pool. Using the relationship δX = (RX/RSt) − 1, where δX is δ15N of component X, RX is 15N/14N of component X, and RSt is 15N/14N of a standard (N2 in air for nitrogen), Equation 6 can be written as:

graphic file with name M7.gif (7)

Subtracting Equation 5 from Equation 7 gives:

graphic file with name M8.gif (8)

Substituting from Equation 5, and from the relationship δRn = δLn − b derived above, Equation 8 can be expressed as:

graphic file with name M9.gif (9)

Expanding Equation 9, canceling terms, and rearranging leads to:

graphic file with name M10.gif (10)

Having assumed that the pool of unreacted nitrate in the plant is negligible compared with the organic pool and that nitrate uptake proceeds at steady state, the ratio of pools NRn/NP can be set equal to the ratio of fluxes FRn/FNn. If we make the further assumption that the nitrogen pool in roots contains only root-assimilated nitrogen, that is to say, that any shoot-assimilated nitrogen transported to the roots in phloem sap does not remain in the roots but is transported back to the shoot, then δRn can be set equal to δR, where δR is the δ15N of root organic material. Applying these assumptions, Equation 10 can be written as:

graphic file with name M11.gif (11)

Finally, combining Equations 4 and 11 yields the following expression for δP:

graphic file with name M12.gif (12)

Equation 12 suggests that the δ15N of plant nitrogen absorbed from the soil as nitrate should vary as a function of three terms, assuming that b is a constant: δSn, FNn/FIn, and δP − δR. For situations where δSn can reasonably be assumed to be similar among plants (e.g. for plants growing at a common site), δP for nitrate-derived plant nitrogen should thus vary as a function of FNn/FIn and δP − δR. The FNn/FIn is the ratio of the net uptake of nitrate by the plant to the influx of nitrate from the soil into the roots. The FIn represents a one-way, or gross, flux. The importance of recognizing one-way fluxes in isotopic modeling has been recently highlighted (Cernusak et al., 2004; Farquhar and Cernusak, 2005). It is necessary to decompose net fluxes into component one-way fluxes, because the one-way fluxes can carry different isotopic signatures.

Dalton et al. (1975) and Fiscus (1975) suggested the following expression for a net solute flux (FNs) across a membrane in the steady state (mol m−2 s−1):

graphic file with name M13.gif (13)

where ω is the permeability coefficient of the membrane (mol m−2 s−1 Pa−1), R is the gas constant (J mol−1 K−1), T is temperature (K), COs is the solute concentration outside the membrane (mol m−3), CIs is the solute concentration inside the membrane (mol m−3), σ is the membrane reflection coefficient (dimensionless), v is the flux of water across the membrane (m3 m−2 s−1), and Fs* is the active uptake of solute across the membrane (mol m−2 s−1). For a charged solute, the active uptake term would include both the metabolically supported transport across the membrane and the flow of ions down the resultant electrochemical gradient (Fiscus and Kramer, 1975). Following this approach, we suggest the following conceptual expression for the one-way flux of nitrate from the soil into root cells (FIn):

graphic file with name M14.gif (14)

where CRn is the nitrate concentration at the root surface (mol m−3), Fn* is the active uptake of nitrate (mol m−2 s−1) as defined above for an ionic solute, and the other terms are as described for Equation 13. Equation 14 expresses the influx of nitrate into root cells as the sum of three component fluxes: a diffusive flux (ωRTCRn), a convective flux [(1 − σ)CRnv], and an active uptake flux (Fn*). Note that this formulation assumes that the three component fluxes operate independently, such that the efflux is not affected by the convective component (Dalton et al., 1975; Fiscus, 1977, 1986). Assuming that efflux occurs as a result of passive leakage of nitrate out of root cells (Peoples and Gifford, 1997; Crawford and Glass, 1998), FEn could then be defined as:

graphic file with name M15.gif (15)

where CCn is the cytosolic nitrate concentration. The efflux would thus result from diffusion of nitrate across the plasmalemma. Equations 14 and 15 represent the component one-way fluxes that combine to give the net solute flux described by Equation 13. Thus, one obtains Equation 13 by subtracting Equation 15 from Equation 14.

We suggest that the influx of nitrate into the roots partly depends upon the flux of water into the roots (v), such that FNn/FIn should correlate with FN/v for plants growing in soil of similar nitrate concentration. The FN is net nitrogen uptake from all nitrogen sources but is assumed to result from only nitrate in this example. Equation 14 indicates that at a given value of CRn, FIn would be expected to increase with increasing v, due to an increase in the convective flux, also referred to as the solvent drag flux (Fiscus, 1975), so long as σ is less than unity. The v is expressed as m3 water m−2 root surface s−1 in Equations 13 and 14. It can also be expressed as mol water m−2 root surface s−1. In this paper, we employ the latter, such that FN/v has units of μmol nitrogen mol−1 water.

In addition to the influence of v on FIn due to the convective component of the influx, the soil solution nitrate concentration at the root surface, CRn, also partly depends upon v. Nitrate can be transported to the root surface both by diffusion and by mass flow. For the simplified case of an isolated root in one dimension in the steady state, CRn can simply be described as CRn = CSnv/α, where CSn is the nitrate concentration of the soil solution outside the disturbance zone associated with the root and α is the root absorbing power, defined as FNn/CRn (Nye and Tinker, 1977). This relationship can be understood by considering the situation where a depletion zone has spread from the root surface outward for some distance into the soil. In the steady state, the flux of nitrate entering the depletion zone from the soil must be the same as the net flux of nitrate leaving the depletion zone at the root surface. Under such conditions, CRn will be a function of the interplay between nitrate supply, represented by CSnv, and root demand for nitrate, represented by α. If the α does not change with changing CRn, CRn will vary as a function of v, assuming constant CSn. As shown in Equation 14, an increase in CRn would be expected to cause an increase in FIn.

To summarize, we suggest that there are two components to the dependence of FIn on v. First, at a given CRn, the convective component of FIn would be expected to increase with increasing v, as shown in Equation 14. Second, CRn partly depends upon v, such that if CRn increases as a function of increasing v, this should also cause an increase in FIn.

The above theoretical considerations lead us to suggest that for plants deriving their nitrogen from soil nitrate and growing in soil with similar CSn and δSn, δP should vary as a function of δP − δR and FN/v, which we term the transpiration efficiency of nitrogen acquisition (Cernusak et al., 2007a). Furthermore, these correlations may hold for plants with multiple nitrogen sources if a significant fraction of plant nitrogen is derived from soil nitrate. In addition, if soil ammonium is the plant's nitrogen source, the same argument can be constructed suggesting that FNa/FIa should correlate with FN/v, where FNa is net uptake of ammonium from the soil and FIa is the influx of ammonium from the soil into root cells.

A full description of the effects of multiple nitrogen sources on plant δ15N can be developed by expanding the above treatment. For plants with multiple nitrogen sources, the following mass balance equation can be written for net uptake of nitrogen:

graphic file with name M16.gif (16)

where FN is net uptake of nitrogen from all nitrogen sources, FNn is net uptake of soil nitrate, FNa is net uptake of soil ammonium, FNd is dinitrogen (N2) fixation from the atmosphere, and FNo is net uptake of organic nitrogen from the soil. Expressing Equation 16 for 15N and applying similar approximations to those applied above leads to the following expression for the δ15N of nitrogen uptake from all four nitrogen sources:

graphic file with name M17.gif (17)

where δSa is the δ15N of soil ammonium, c is the discrimination constant for assimilation of ammonium by Gln synthetase, estimated to be about 17‰ (Yoneyama et al., 1993), ΔNd is discrimination against 15N during N2 fixation, which ranges from approximately 0‰ to 2‰ (Yoneyama et al., 1986), δSo is δ15N of the soil organic nitrogen pool available to the plant, and ΔNo is discrimination during net uptake of soil organic nitrogen. Little is known about ΔNo, but it appears to vary depending on the type of organic molecule taken up (Schmidt et al., 2006). Equation 17 assumes that nitrate and ammonium assimilation proceed independently and that atmospheric N2 has a δ15N of 0‰ (Mariotti, 1983). Free ammonium is generally not detected in xylem sap, except in trace amounts (Peoples and Gifford, 1997), so all ammonium assimilation is assumed to take place in roots.

For plants incapable of dinitrogen fixation and taking up little or no nitrogen as organic molecules from the soil, Equation 12 may provide a reasonable approximation to Equation 17, in that positive relationships between δP and FN/v and between δP and δP − δR could be maintained, despite variable uptake of soil nitrogen as nitrate versus ammonium. We tested the generality of these predictions using a diverse suite of tropical tree and liana species. To provide further insight into the effects of variable uptake of nitrate versus ammonium on the predicted relationship between δP and FN/v, we conducted a sensitivity analysis using Equation 17 and assuming a range of values for FNn and FNa.

RESULTS

Figure 2 shows correlations between δP and FN/v and between δP and δP − δR for 15 species of tropical trees and lianas, the former including both conifers and angiosperms. These plants were grown individually in 38-L pots under well-watered conditions at a Smithsonian Tropical Research Institute field site in the Republic of Panama. Soil was homogenized at the beginning of the experiment, and the bulk soil had a δ15N of 5.1‰. We observed positive correlations between both δP and FN/v (Fig. 2A) and between δP and δP − δR (Fig. 2B). The dashed lines in Figure 2 represent least-squares linear regressions. The linear regression in Figure 2A does not include the species Platymiscium pinnatum, a leguminous tree species that formed nitrogen-fixing nodules on its roots. Fixation of atmospheric nitrogen would be expected to alter the correlation between δP and FN/v, both by increasing FN/v and by shifting δP toward 0‰. Such a trend can be seen for P. pinnatum in relation to the correlation for the other 14 species shown in Figure 2A.

Figure 2.

Figure 2.

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δP plotted against FN/v (A) and δP − δR (B). Dashed lines represent least-squares linear regressions. Regression equations and statistics are given in each panel. The regression analysis in A does not include the leguminous tree species P. pinnatum, represented by black hexagons, which are enclosed in gray circles to make them more discernible. This species forms N2-fixing nodules on its roots, which is expected to alter δP through an additional process not present in the other species. White symbols with internal cross-hairs refer to conifer tree species; completely white symbols refer to angiosperm liana species; black symbols and black symbols with internal cross-hairs refer to angiosperm tree species.

Table I summarizes a multiple regression analysis of the data set presented in Figure 2, with δP as the dependent variable and FN/v and δP − δR as independent variables. The regression model explained 55% of variation in δP, and both FN/v and δP − δR were significant terms in the model (Table I). The standardized coefficients indicate that FN/v was a slightly stronger term than δP − δR. P. pinnatum was excluded from the analysis presented in Table I. With P. pinnatum included in the analysis, the regression model explained 66% of variation in δP, with most of the increase in explanatory power attributed to FN/v.

Table I.

Multiple regression analysis with δP as the dependent variable and FN/v and δP − δR as independent variables

Data included in the analysis are plotted in Figure 2, which also gives the species identities. The analysis shown here did not include the leguminous tree species P. pinnatum, which formed N2-fixing nodules on its roots. For the analysis, r2 = 0.55, F = 52.0, P < 0.001, and n = 89.

Independent Variable Coefficient se Standardized Coefficient P
Constant −0.521 0.325 0.11
FN/v (μmol nitrogen mol−1 water) 0.094 0.013 0.537 <0.001
δP − δR (‰)
 0.963
 0.156
 0.452
 <0.001
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Figure 3 shows an example of the interplay between δP − δR and FN/v in determining δP for two tropical tree species, Tectona grandis and Swietenia macrophylla. Five seedlings of each species were grown individually in 19-L pots under well-watered conditions in homogenously mixed, unfertilized forest topsoil. Both T. grandis and S. macrophylla showed positive correlations between δP and δP − δR (Fig. 3A). The offset between species in these correlations was explained by variation in FN/v (Fig. 3B). Results of a multiple regression analysis for the data presented in Figure 3 are shown in Table II. For this data set, the multiple regression model explained 91% of variation in δP, with both δP − δR and FN/v being significant terms. As in the analysis of data presented in Figure 1, FN/v was a slightly stronger term in the model than δP − δR, as can be seen from the standardized coefficients for the two independent variables (Table II).

Figure 3.

Figure 3.

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A, δP plotted against δP − δR for five individuals each of two angiosperm tree species, T. grandis and S. macrophylla. B, Variation between the two species in FN/v of the same plants. Dashed lines in A are least-squares linear regressions. Error bars in B represent 1 sd.

Table II.

Multiple regression analysis with δP as the dependent variable and FN/v and δP − δR as independent variables

The data set comprised five individuals each of T. grandis and S. macrophylla. Data included in the analysis are plotted in Figure 3. For the analysis, r2 = 0.91, F = 37.3, P < 0.001, and n = 10.

Independent Variable Coefficient se Standardized Coefficient P
Constant −1.047 1.280 0.44
FN/v (μmol nitrogen mol−1 water) 0.232 0.031 0.915 <0.001
δP − δR (‰)
 1.282
 0.182
 0.861
 <0.001
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DISCUSSION

Application of a theoretical model of nitrogen isotope fractionation during nitrate uptake (Comstock, 2001) led us to predict that δP would correlate with FN/v and δP − δR for plants grown in a common soil environment. We tested these hypotheses with measurements of δP in tropical tree and liana seedlings grown in homogenized soil under well-watered conditions. In agreement with theoretical predictions, results suggested that both FN/v and δP − δR were significant sources of variation in δP.

In addition to influences on δP associated with uptake of nitrate, Equation 17 clearly indicates the potential for influences associated with uptake of soil ammonium, uptake of soil organic nitrogen, and fixation of atmospheric nitrogen. The influence of the latter can be seen in Figure 2A in relation to P. pinnatum, as noted above. In addition, it is possible that some of the unexplained variation in the multiple regression analysis presented in Table II could have resulted from variation among species in preference for ammonium versus nitrate, or possibly from uptake of organic nitrogen from the soil. The importance of organic nitrogen uptake in woody tropical plants is largely unknown (Näsholm et al., 2009), although a capacity for uptake of Gly has been demonstrated in a small number of such species (Schmidt and Stewart, 1999; Wanek et al., 2002). On the other hand, woody tropical plants show a range of preferences for uptake of nitrate relative to ammonium (Stewart et al., 1988; Schmidt and Stewart, 1999; Arndt et al., 2002; Wanek et al., 2002; Aidar et al., 2003; Schimann et al., 2008).

Assuming soil nitrate and soil ammonium as available nitrogen sources for a non-N2-fixing plant, Equation 17 indicates that variation in δP could result from differences in the proportion of FN accounted for by FNn versus FNa, from differences between δSn and δSa, and from differences in discriminatory processes associated with the two net fluxes. Variation in the δ15N of leaves caused by variation in FNn relative to FNa is consistent with some recent analyses (Garten, 1993; Miller and Bowman, 2002; Falkengren-Grerup et al., 2004; Houlton et al., 2007; Kahmen et al., 2008), and the potential for discrimination associated with nitrate and ammonium uptake is well recognized (Mariotti et al., 1982; Handley and Raven, 1992; Robinson et al., 2000; Evans, 2001; Yoneyama et al., 2001; Kolb and Evans, 2003; Pritchard and Guy, 2005).

A sensitivity analysis of the impact of preference for uptake of nitrate versus ammonium on the relationship between δP and FN/v is shown in Figure 4A. The key assumptions underlying the relationships shown in Figure 4 are that FNn/FIn and FNa/FIa are linearly correlated with FNn/v and FNa/v, respectively. If these assumptions hold true, Figure 4 indicates that the positive relationship between δP and FN/v is relatively robust in the face of a variable preference for nitrate versus ammonium uptake. Altering the proportion of FN accounted for by FNn versus FNa (Fig. 4A), the partitioning of nitrate assimilation between root and shoot (Fig. 4B), or the difference between δSn and δSa (Fig. 4C) can lead to moderate variation in the slope and/or intercept of the predicted relationship between δP and FN/v; however, the positive correlation between the two parameters remains.

Figure 4.

Figure 4.

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A sensitivity analysis of the predicted relationship between whole-plant δ15N and FN/v as a function of the proportion of nitrogen uptake from the soil as nitrate versus ammonium (A), the proportion of nitrate assimilation in roots versus leaves (B), and the δ15N difference between soil nitrate and soil ammonium (C). Equation 17 was used to predict whole-plant δ15N, assuming no fixation of atmospheric N2 and no uptake of organic nitrogen from the soil (i.e. FNd = 0, FNo = 0). Ratios of net flux to influx for soil nitrate and ammonium were assumed to correlate linearly with the transpiration efficiency of nitrogen acquisition for each nitrogen source, according to the following relationships: (FNn/FIn) = (FNn/v)/100 and (FNa/FIa) = (FNa/v)/100. The range of parameter values considered for each analysis is given above each panel. If a parameter value is not given above the panel, the following were assumed: (FNn/FN) = 0.5, (FNa/FN) = 0.5, b = 15‰, c = 17‰, (FRn/FNn) = 0.5, δSn = 3‰, and δSa = 7‰.

Discrimination against 15N during uptake of either nitrate or ammonium can occur when there is a significant efflux of that ion from root cells. Significant variation has been observed among species in efflux-to-influx ratios for both nitrate (1 − FNn/FIn) and ammonium (1 − FNa/FIa) (Min et al., 1999; Scheurwater et al., 1999; Britto et al., 2001; Kronzucker et al., 2003). In general, 1 − FNn/FIn and 1 − FNa/FIa appear to increase with increasing concentration of nitrate or ammonium, respectively, in the rooting solution (Teyker et al., 1988; Siddiqi et al., 1991; Wang et al., 1993; Glass, 2003; Britto and Kronzucker, 2006). This supports the suggestion that an increase in nitrate or ammonium concentration at the root surface caused by an increase in v should also lead to an increase in 1 − FNn/FIn or 1 − FNa/FIa. Because nitrogen availability is generally assumed to be higher in tropical than in temperate forests (Vitousek and Howarth, 1991; Houlton et al., 2008), discrimination associated with efflux of nitrate and ammonium from root cells may be more pronounced in tropical than in temperate trees.

Equation 17 indicates that in the case of nitrate the possibility for discrimination associated with efflux from root cells can be completely canceled if all nitrate reduction takes place in the shoot rather than in roots. This is because nitrate loading into the xylem is assumed to be a nondiscriminating process. In this situation, even if there was significant efflux of nitrate from root cells, it would not be recorded in δP. Plants show a variable partitioning of nitrate assimilation between roots and shoots (Andrews, 1986; Stewart et al., 1993), and this is likely to cause variation in δP (Fig. 4B). Results presented in Figure 3A can be interpreted to suggest that S. macrophylla displayed a greater variability among individuals in FRn/FNn than T. grandis, because the former species showed a larger range of δP − δR than the latter.

Intriguingly, some of the highest δP values in the data set shown in Figure 2 were for the potentially N2-fixing species P. pinnatum. Symbiotic N2 fixation in legumes may show a slight discrimination against 15N, in the range of 0‰ to 2‰ (Yoneyama et al., 1986), such that plant nitrogen derived from this source should have a δ15N in the range of approximately 0‰ to −2‰. Roots of the P. pinnatum plants shown in Figure 2A were nodulated, so some degree of N2 fixation seems likely. As noted previously, N2 fixation should shift the relationship between δP and FN/v by pulling δP toward 0‰ and increasing FN/v compared with values for non-N2-fixing plants. Consistent with this suggestion, the relationship between δP and FN/v appears to differ for P. pinnatum than for the rest of the species in Figure 2A, although the difference is admittedly rather subtle. A more dramatic illustration of the difference in relationships between δP and FN/v for N2-fixing, leguminous tree species compared with non-N2-fixing tree species is shown in Figure 5, based on a previously published data set (Cernusak et al., 2007a). A multiple regression analysis for the non-N2-fixing species in Figure 5 is shown in Table III. Results are similar to those shown in Table I. Figure 5 clearly shows a negative slope between δP and FN/v for the leguminous tree species P. pinnatum and Dalbergia retusa, whereas the slope for the non-N2-fixing species is positive.

Figure 5.

Figure 5.

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δP plotted against FN/v for seven tropical tree species. White symbols refer to leguminous tree species capable of forming N2-fixing nodules on their roots, and black symbols refer to tree species incapable of N2 fixation. The solid line and the dashed line represent least-squares linear regressions for non-N2-fixing species and N2-fixing species, respectively. Data presented in this figure were originally published by Cernusak et al. (2007a).

Table III.

Multiple regression analysis with δP as the dependent variable and FN/v and δP − δR as independent variables

The analysis includes data for the non-N2-fixing species shown in Figure 5 (r2 = 0.49, F = 13.2, P < 0.001, and n = 31).

Independent Variable Coefficient se Standardized Coefficient P
Constant 0.422 0.560 0.46
FN/v (μmol nitrogen mol−1 water) 0.041 0.012 0.468 <0.01
δP − δR (‰)
 0.547
 0.157
 0.473
 <0.01
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The intersection of the regression lines in Figure 5 for the leguminous tree species and the non-N2-fixing tree species suggests that in the absence of N2 fixation, the leguminous tree species would have δP values near the highest observed for the non-N2-fixing species. In combination with Figure 2, these data suggest that the leguminous tree species examined may have low rates of nitrate and ammonium efflux from root cells, associated with a high plant demand for nitrogen. This would be consistent with the nitrogen-demanding lifestyle that characterizes legumes more generally (Mckey, 1994).

The theoretical argument put forward in the introduction to explain the correlation between δP and FN/v is conceptually consistent with existing paradigms concerning 15N discrimination during nitrogen uptake. It is generally assumed that whole-plant δ15N is unlikely to show significant discrimination with respect to soil nitrogen sources when plant demand for nitrogen strongly exceeds nitrogen supply, and opportunities for discrimination increase as soil nitrogen supply increases relative to plant nitrogen demand (Evans, 2001). For plants growing in a common soil environment, FN/v is an expression of nitrogen demand relative to nitrogen supply. At steady state, for a given FN, an increase in v will increase the concentration of nitrogenous solutes at the root surface and their transport into the roots (assuming σ < 1), thus increasing nitrogen supply. Similarly, for a fixed FN, a decrease in v would decrease the concentration of nitrogenous solutes at the root surface and the convective flux of such solutes into the roots (assuming σ < 1), thus decreasing supply relative to demand. It is worth pointing out that a weaker relationship would be expected between δP and FN/v for plants grown in hydroponics compared with soil, because solute concentrations at the root surface in a well-stirred hydroponic solution are largely uncoupled from variation in FN/v.

It should be emphasized that a positive correlation between δP and FN/v among a population of non-N2-fixing plants would only be expected when the plants are exposed to inorganic soil nitrogen pools of similar concentration and isotopic composition. If the plants are growing in differing soils in which the nitrate and ammonium concentrations in the soil solution are very different, nitrate and ammonium concentrations at the root surface will vary independently of variation in FN/v. As a consequence, nitrate and ammonium efflux-to-influx ratios will also be uncoupled from FN/v, as will discrimination during nitrogen uptake. This situation can be demonstrated by an analysis of the data set presented by Cernusak et al. (2007b). In that study, seedlings of a tropical pioneer tree (Ficus insipida) were grown in soil containing variable proportions of rice husks, an organic substrate with a high carbon-nitrogen ratio and therefore expected to encourage microbial immobilization of soil nitrogen. Nitrogen availability differed strongly among the treatments, and as a result, no correlation was observed between δP and FN/v (P = 0.12, n = 24).

It has been suggested that root symbioses with mycorrhizas can influence δP (Handley et al., 1993; Högberg, 1997; Hobbie et al., 2000; Schmidt et al., 2006). Most of the tropical tree and liana species examined in this study were likely associated with arbuscular mycorrhizal fungi (Wang and Qiu, 2006), with the exception of Pinus caribaea, which could have supported ectomycorrhizas (Tedersoo et al., 2007). Nonetheless, we are unable to draw any conclusions about the influence, or lack thereof, of mycorrhizal status on δP in our experiment, because we do not have information on mycorrhizal infection levels. However, it will likely prove useful in future work to incorporate a mycorrhizal compartment into the modeling framework for δP. This could be accomplished using the generalized approach for isotope reactions in branched pathways presented by Comstock (2001), assuming that appropriate information about fractionations associated with nitrogenous fluxes between mycorrhizas and roots becomes available.

Interpretation of plant δ15N in ecological investigations is often a complex exercise (Robinson, 2001). It can be simplified if the assumption can be made that mineral nitrogen sources are assimilated without discrimination during uptake, as might be expected when plant demand for nitrogen strongly exceeds nitrogen supply to roots (Evans, 2001). We interpret the positive correlations between δP and FN/v shown in Figures 2, 3, and 5 for non-N2-fixing tropical tree and liana species as resulting from variation in efflux-to-influx ratios for nitrate and ammonium from root cells. We provide a theoretical justification in the introduction for this interpretation. The implication is that, for tropical forest trees and lianas, it cannot simply be assumed that nitrate and ammonium will be taken up from the soil without discrimination against 15N. Moreover, we observed a range of δP values among species (Fig. 2A), which suggests significant variation in mineral nitrogen efflux rates from roots. Such variation could have important implications for ecosystem nitrogen cycling and interspecific competition (Glass, 2003; Kronzucker et al., 2003), because it suggests that tropical tree and liana species vary in their abilities to rapidly absorb available nitrogen from the soil.

CONCLUSION

We have presented experimental evidence from several tropical tree and liana species in support of the hypothesis that variation in δP should correlate with variation in FN/v and δP − δR for plants grown in a similar soil environment. Furthermore, we have demonstrated contrasting behavior in the relationship between δP and FN/v in non-N2-fixing compared with N2-fixing species, also consistent with theoretical expectations. These results make a novel contribution to the theoretical framework that can be brought to bear upon the interpretation of δ15N variations in terrestrial plants.

MATERIALS AND METHODS

Experiments were carried out at the Santa Cruz Experimental Field Facility, Smithsonian Tropical Research Institute, Republic of Panama (9°07′ N, 79°42′ W), at an altitude of approximately 28 m above sea level. For data presented in Figure 2, a full account of plant growth conditions and experimental procedures was given previously (Cernusak et al., 2008). Briefly, plants were grown for several months individually in 38-L pots that did not drain. Cumulative plant water use over the experimental period was determined by weighing the pots at weekly or subweekly intervals and replacing the water consumed in the preceding interval. Soil evaporation was estimated by weighing control pots that contained no plants. At harvest, plants were separated into roots, stems, and leaves and dried to constant mass at 70°C. Plant material was ground to a fine powder for elemental and isotopic analyses. The δ15N and nitrogen concentrations of dry matter were determined on subsamples of approximately 3 mg that were combusted in an elemental analyzer (ECS 4010; Costech Analytical Technologies) coupled to a continuous flow isotope ratio mass spectrometer (Delta XP; Finnigan MAT) at the Stable Isotope Core Laboratory, Washington State University. The FN/v was calculated as [(MF − MI)NF]/ET, where MF and MI are final and initial dry mass of the plants, respectively, NF is the whole-plant nitrogen mass fraction at final harvest, and ET is cumulative transpiration. Thus, the initial nitrogen mass fraction was assumed to be equal to that at harvest. The mean MI was estimated as 1.8 g, and the mean MF was 50.1 g, such that any variation in initial nitrogen concentration would have had a negligible influence on estimates of FN/v.

The experimental procedure for measurements shown in Figure 3 was similar to that described above, except that plants were grown in 19-L pots. Full experimental details, including descriptions of growth and physiology, are provided elsewhere (Cernusak et al., 2009). The δ15N data for data sets presented in Figures 2 and 3 are presented for the first time in this paper. Statistical analyses were performed in SYSTAT 11.0 (SYSTAT Software).

Acknowledgments

We thank Ben Harlow and Dayana Agudo for assistance with isotopic analyses and Jorge Aranda, Milton Garcia, and Aurelio Virgo for technical assistance with experiments.

1

This work was supported by a Tupper Postdoctoral Fellowship from the Smithsonian Tropical Research Institute and by an Australian Postdoctoral Fellowship from the Australian Research Council, both to L.A.C.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Lucas A. Cernusak (lucas.cernusak@cdu.edu.au).

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