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Ecology |
Department of Plant Biology, Cornell University, Ithaca, New York 14855-5908 USA
Received for publication December 23, 2004. Accepted for publication April 11, 2005.
ABSTRACT
We report the nitrogen (N), phosphorus (P), and carbon (C) stoichiometry for each of the five organ-types (leaves, aerial stems, reproductive organs, roots, and tubers) of 17 actively growing Eranthis hyemalis plants differing in size (as measured in g C). We also report the N, P, and C stoichiometry of 20 winterized tubers, which are the only perennial organs of this species. Comparisons between whole-plant and winterized N/C and P/C levels indicate that N was resorbed from aerial organs and stored in tubers by the end of the growing season. Leaves were substantial reservoirs for N and P. With few exceptions, N scaled isometrically with respect to C for each organ-type, whereas P scaled as the 3/4 power of C. Thus, N is proportional to P3/4, which is proportional to C regardless of organ-type. Additionally, annual growth rate G of shoots (leaves and aerial stems) scaled as the –3 power of leaf N/P quotients such that G was proportional to the 3/4 power of leaf P. We suggest that these scaling relationships (together with previously reported allometric trends across herbaceous species) show that growth is constrained by organ-specific N and P allocation patterns (presumably to proteins and ribosomes, respectively).
Key Words: allocation patterns • essential nutrients • nitrogen • phosphorus • protein-rRNA growth models
Body or organ sizes are typically reported in units of dry mass or, less commonly, in units of carbon mass in the majority of plant allometric studies (e.g., Goodall, 1945
, 1950
; Beets, 1980
; Niklas, 1994
; Niklas and Enquist, 2001
). However, neither of these measures has intrinsic priority over alternatives, some of which can provide deeper insights into the allometry of growth, development, or reproductive success. For example, nitrogen (N) is an essential constituent of amino acids, amides, nucleic acids, nucleotides, coenzymes, hexamines, and many other carbon containing compounds, whereas phosphorus (P) is an essential constituent of ribosomes, ATP, sugar phosphates, and phospholipids. Thus, tissue or organ N levels provide a reasonable gauge of the protein "overhead" that must be synthesized to maintain balanced growth, whereas P levels offer a crude gauge of the "machinery" driving growth (Sterner and Elsar, 2002
; Ågren, 2004
; Vrede et al., 2004
).
Importantly, N and P tissue levels vary among foliage leaves, stems, and roots as a consequence of structural and physiological differences. These levels also change as a function of annual mobilization from storage to growing sites within the plant body. The organographic distribution and phenology of essential nutrients are thus intimately linked to the allometry of growth and development. For example, across the few woody species previously examined, leaf N concentrations are reported to be higher than those in roots (Kozlowski, 1971
; Dyck et al., 1988
) largely because of significant amounts of ribulose bisphosphate carboxylase/oxygenase (Rubisco) in photosynthetic organs (Calvin cycle and thylakoid proteins contain the majority of leaf nitrogen; see Evans, 1989
; Zhang et al., 2003
). Likewise, N and P are recruited from older branches and mobilized in swelling buds during the early growing season (e.g., Mochizuki and Hanada, 1958
; Meyer and Tukey, 1965
; Taylor, 1967
; Taylor and May, 1967
). And, on average, 50% of the N and P in green leaves is reabsorbed during senescence; between 82% and 91% of the resorbed N comes from the hydrolysis of proteins, whereas 50% of the resorbed P comes from nucleic acid hydrolysis (Chapin and Kedrowski, 1983
; Aerts, 1996
).
Nevertheless, few studies report intra- or interspecific size-dependent variations in leaf, stem, and root N and P concentrations with respect to carbon (C) content. The objective of this study was to examine this allometry for Eranthis hymalis (L.) Salisb. (Ranunculaceae), a perennial herbaceous species that overwinters by means of tubers. This species was selected for two primary reasons: N or P recruitment can only involve transport to or from tubers (because field observations indicate that tubers are the only organs that persist throughout the winter), and because secondary tissues in tubers are minimal (such that N or P sequestration in the walls of nonliving cells is minimal).
MATERIALS AND METHODS
Seventeen actively growing plants (five with developing green fruits and seeds) with intact stems and leaves evincing little or no insect or microbial damage were collected during the summer of 2004 (Fig. 1). These specimens were selected in part because of their size-range (which enhanced the probability that allometric trends could be detected). Roots were excavated by removing soil balls with diameters and depths measuring at least twice the height of the tallest aerial stem of each plant. Soil balls were placed in containers and washed repeatedly until the roots were exposed fully. Any remaining soil was subsequently removed from roots using dissecting needles with the aid of a dissecting microscope. Shoots were also washed to remove any attached soil or debris.
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Twenty tubers were also collected during the winter of 2003 to determine N and P storage levels. Tubers were collected because they are the only winterized organs (field observations indicate that all roots are absent from winterized tubers).
Each of the 17 growing plants was dissected into its five organ-types (leaves, aerial stems, reproductive organs, roots, and tubers), which were subsequently air dried on laboratory benches for a minimum of 2 weeks to reduce as much as possible microbial decomposition. The tubers of all 37 plants were sliced open to expedite drying. Each was weighed periodically to determine whether the other organs from the same plant had dried completely. The dry mass (in g) of each organ-type of each plant was measured before determining N, P, and C levels. Carbon mass was used as the measure of organ or whole-plant size.
Nitrogen tissue content was evaluated as total Kjeldahl N. After oven drying (70°C), each sample was finely ground and powdered and then combusted at 1800°C. Copper was used as a reducing agent to remove O2 and to reduce a sample to its elemental nitrogen, carbon dioxide, and water. The combustion effluent of each sample was then analyzed chromatographically for nitrogen and carbon dioxide using a semi-automated procedure. Using helium as the carrier gas, the chromatographic effluent for each sample was first passed through a water trap (to remove water). Atropine standards (0.5–4.0 mg) were used to calibrate each analysis. The nitrogen and carbon readings for each sample fell within 0.5% of the comparable readings of any given atropine standard. Carbon, nitrogen, and hydrogen composition was also determined using an elemental analyzer (Carlo Erba, Milano, Italy).
Phosphorus tissue content was determined using nitric/sulfuric acid digestion and vanadomolybdophosphoric (or ascorbic) acid methods (Wilde et al., 1972
; Pierzynski, 2000
). Briefly, 1 mL of concentrated H2SO4 and 5 mL of concentrated HNO3 were added to a known volume of a well-mixed, dried tissue sample, which was subsequently digested to a volume of 1 mL and further until the solution was colorless (to remove HNO3). The resulting solution was cooled and diluted with 20 mL of distilled water. A phenolphthalein indicator and 1 molar NaOH solution were used to neutralize this solution (as gauged by a faint pink color). When necessary, the neutralized solution was filtered to remove particulates (sometimes observed for large tubers). Depending on preliminary analyses of duplicate samples, either the vanadomolybdophosphoric acid or the ascorbic acid colormetric method was used to determine P in solution (samples for which 1 < mg P/L 
20 or 0.01 < mg P/L 6, respectively).
Standard all-pairwise t-test comparisons (
= 0.05) were used to determine whether mean N, P, N/C, or P/C values differed statistically across the different organ-types. Model type II (reduced major axis, RMA) regression analyses were used to determine the scaling exponents (i.e., the slopes of RMA regression curves, denoted as RMA) for N vs. C and P vs. C because functional (rather than predictive) relationships were sought. It should be noted, however, that the slopes of RMA regression curves and those of ordinary least squares (Model type I) regression curves differ significantly only when r2 < 0.95 (see Niklas, 1994
).
RESULTS
The absolute mass of N and P (and the mass of N and P normalized with respect to that of carbon per organ) differed significantly among the five organ-types (Fig. 2). In terms of mean ± SE of N or P levels in each organ-type, reproductive organs and winterized tubers were, on average, the largest reservoirs for N and P (i.e., reproductive organs = 0.0033 ± 0.002 g N and 0.0003 ± 0.001 g P; tubers = 0.0054 ± 0.004 g N and 0.0021 ± 0.001 g P), whereas roots and aerial stems, on average, are N- and P-poor (i.e., root mean ± SE = 0.0004 ± 0.001 g N and 0.0004 ± 0.001 g P; aerial stems mean ± SE = 0.0005 ± 0.001 g N and 0.0006 ± 0.001 g P) (Fig. 2A–B). However, when normalized with respect to their carbon content (absolute size), leaves were N-rich (i.e., mean N/ C ± SE = 0.0465 ± 0.002), whereas the tubers of growing plants were N-poor (i.e., mean N/C ± SE = 0.0075 ± 0.002) (Fig. 2C). Likewise, roots were P-rich (i.e., mean P/C ± SE = 0.0298 ± 0.011) and leaves were P-poor (i.e., mean P/C ± SE = 0.0049 ± 0.003) (Fig. 2D).
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Finally, we note that annual shoot growth decreases with increasing leaf N/P quotients. Specifically, across the 17 growing plants, the scaling exponent for annual growth vs. N/P was –2.80 ± 0.06 (95% CI = –2.98 to –2.73; r2 = 0.993, F = 2,206, P < 0.0001) (Fig. 5A). Assuming that this exponent is approximated numerically by –3, we see from the proportional relationships N 
P3/4 and G (N/P)–3 that G should scale as the 3/4 power of P. This prediction was substantiated empirically, i.e., the scaling exponent for G vs. P was 0.76 ± 0.02 (95% CI = 0.71–0.81; r2 = 0.986, F = 1,087, P < 0.0001) (Fig. 5B).
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The N and P stoichiometry of Eranthis hyemalis illustrates a number of phenomena that have been reported individually for different species. But to our knowledge, all of these phenomena have not been reported for a single species and thus integrated within the context of a single taxon. Among these phenomena are (1) significant differences in the absolute (as well as the C-normalized) amounts of N and P allocated to different organ-types (Thompson et al., 1997
), (2) comparatively high concentrations of N in photosynthetic organs (Evans, 1989
; Reich and Oleksyn, 2004
), (3) the resorption of N and P from perennial organs during early growth and the storage of these essential nutrients in winterized organs (Meyer and Tukey, 1965
; Chapin and Kedrowski, 1983
; Averts, 1996
; Zhang et al., 2003
.), and (4) the decrease in annual growth rates with increasing levels of N/P (provided that growth rates are not extremely slow; Ågren, 2004
; Güsewell, 2004
), which can now be placed in the context of our observation that, across E. hyemalis leaves, N P3/4.
The differences in N/C and P/C quotients among the different organ-types reported for E. hyemalis (and by other workers for other species, e.g., Mochizuki and Hanada, 1958
; Meyer and Tukey, 1965
; Chapin and Kedrowski, 1983
; Niklas et al., 2005
) can be ascribed to differences in organ-specific N and P allocation patterns to proteins and P-rich cellular components, the cycling of N and P among different organs during growth and senescence, and to organ-specific differences in the accumulation of storage carbohydrates (Dyck et al., 1988
; Averts, 1996
; Sterner and Elser, 2002
; Güsewell, 2004
). For example, the high N/C quotients we report for the leaves of E. hyemalis appear to be the result of a substantial N allocation to Rubisco. For this species, preliminary analyses indicate that 
30% of total leaf mass consists of proteins of which Rubisco is 60%. Assuming that N comprises 16% of the mass of proteins like Rubisco, the N allotted to Rubisco is, on average, 2.9% of total leaf N content of which a large portion (i.e., 40%) resides in chloroplast thylakoids (presumably in the form of pigment–protein complexes and components of the electron transport chain; see Sterner and Elser, 2002
). In passing, we note that ambient light conditions (which can change seasonally for early emerging species like E. hyemalis) also influence N allocation to Rubisco and chloroplast thylakoids (see Evans, 1989
; Zhang et al., 2003
).
Likewise, N and P recruitment from one organ-type and translocation to another type, in tandem with the hydrolysis of storage carbohydrates (e.g., starch), can also alter N/C and P/ C seasonally (see Mochizuki and Hanada, 1958
; Meyer and Tukey, 1965
; Taylor, 1967
; Taylor and May, 1967
). For example, Chapin and Kedrowski (1983)
and Aerts (1996)
have shown that 50% of the N and P in the green leaves of woody species is resorbed during senescence (82% and 91% of resorbed N comes from the hydrolysis of proteins, whereas 50% of resorbed P comes from nucleic acid hydrolysis). Nitrogen resorption from E. hyemalis shoots likely accounts for the increase in tuber N/C toward the end of the growing season (see Fig. 3A).
However, despite all of these organographic and phenological differences in N and P organ levels, we have shown that the allometry of N and P with respect to C is predictable and consistent among the physiologically distinct organ-types of E. hyemalis. For this species, N typically scales isometrically with respect to organ C, whereas P scales as the 3/4 power of C such that, in proportional terms N P3/4. We believe that this scaling relationship is not unique to E. hyemalis and that it has potentially important implications to our definitive understanding of plant growth. Other workers have reported data that comply with the N P3/4 relationship. For example, using the data reported by Cornelissen et al. (1997)
for a broad spectrum of species, Güsewell (2004)
provides a bivariate plot of leaf N vs. leaf P and reports that the slope of the ordinary least squares regression curve is 1.20 with r = 0.85 (Fig. 7b of Güsewell, 2004
). Although Güsewell does not provide the 95% confidence intervals for this regression curve, the slope of the corresponding reduced major axis regression curve is 1.20/0.85 = 1.41, which indicates that P disproportionately increases as N increases in a manner that is consistent with the trend we report for E. hyemalis. Likewise, reanalysis of the data reported by Reich and Oleksyn (2004)
indicates that N P3/4 for leaves of species occupying many different biomes (P. Reich, University of Minnesota, personal communication).
If the proportional stoichiometric relationship N P3/4 holds true across ecologically and physiologically diverse species as well as within individual species (as is suggested by our data and those reported by other workers), then allometric theory obtains the prediction that the annual growth rates G of these species must decrease as the –3 power of N/P levels in leaves and that it must increase as the 3/4 power of leaf P levels. This follows from prior work, which shows that annual growth G scales isometrically with respect to standing leaf mass ML across herbaceous species, i.e., G ML (Niklas and Enquist, 2001
), and from our data, which indicates that standing leaf mass scales isometrically with respect to leaf carbon content (i.e., C ML) and that leaf carbon content scales isometrically with N and as the 3/4 power of P (i.e., C N P3/4). Collectively, these trends indicate that G N P3/4 such that G (N/P)–3.
These expectations are substantiated empirically for E. hyemalis. Across the 17 plants with actively growing shoots, annual shoot growth scaled approximately as the –3 power of leaf N/P quotients and as the 3/4 power of leaf P levels (see Fig. 5). Although we cannot allege that N and P levels constrain growth across all species, there are good reasons to believe that growth depends on N and P allocation patterns (to proteins and ribosomal RNA, respectively) for both plants and animals (see Sterner and Elsar, 2002
; Ågren, 2004
; Vrede et al., 2004
; Niklas et al., 2005
). Indeed, this perspective is mathematically embedded in a host of protein-rRNA "tradeoff" models for growth that view N tissue levels as surrogate measures of the protein "overhead" that must be maintained to achieve balance growth and that judge P tissue levels as crude estimates of the ribosomal "machinery" driving growth. Such models have been very successful in predicting the growth of unicellular and multicellular plant and animal species, which suggest their canonical biological application (see Vrede et al., 2004
; Niklas et al., 2005
).
The data and allometric suppositions we present here amplify (but do not draw) on these models. Certainly, the N P3/4 scaling relationship indicates, at least in theory, that the rRNA "machinery" in leaves may increase disproportionately as the protein "overhead" in the principal light-harvesting organ increases. If true, this may explain why annual growth rates in body mass tend to decline as the 3/4 power of body mass across a broad spectrum of photoautotrophic species (see Niklas, 1994
; Brown and West, 2000
; Niklas and Enquist, 2001
).
FOOTNOTES
1 Support from the College of Agriculture and Life Sciences of Cornell University is gratefully acknowledged. 
2 Author for correspondence (e-mail: kjn2{at}cornell.edu
), phone 607-255-8727, fax 607-255-5407
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