Intra‐trophic isotopic discrimination of 15N/14N for amino acids in autotrophs: Implications for nitrogen dynamics in ecological studies

Abstract The differential discrimination of nitrogen isotopes (15N/14N) within amino acids in consumers and their diets has been routinely used to estimate organismal tropic position (TP). Analogous isotopic discrimination can occur within plants, particularly in organs lacking chloroplasts. Such discrimination likely arises from the catabolic deamination of amino acids, resulting in a numerical elevation of estimated TP, within newly synthesized biomass. To investigate this phenomenon, we examined the 15N/14N of amino acids (δ15 NAA) in spring leaves and flowers from eight deciduous and two annual plants. These plants were classified on the basis of their time of bloom, plants that bloomed when their leaves were absent (Type I) versus plants that bloomed while leaves were already present (Type II). Based on the δ15 NAA values from leaves, both plant types occupied comparable and ecologically realistic mean TPs (=1.0 ± 0.1, mean ± 1σ). However, the estimated TPs of flowers varied significantly (Type I: 2.2 ± 0.2; Type II: 1.0 ± 0.1). We hypothesize that these results can be interpreted by the following sequence of events: (1) Type I floral biomass is synthesized in absence of active photosynthesis; (2) the catabolic deamination of amino acids in particular, leaves behind 15N in the residual pool of amino acids; and (3) the incorporation of these 15N‐enriched amino acids within the biomass of Type I flowers results in the numerical elevation of the TPs. In contrast, the actively photosynthesizing Type II leaves energetically sustain the synthesis of Type II flower biomass, precluding any reliance on catabolic deamination of amino acids. Amino acids within Type II flowers are therefore isotopically comparable to the Type II leaves. These findings demonstrate the idiosyncratic nature of the δ15 NAA values within autotrophic organs and have implications for interpreting trophic hierarchies using primary producers and their consumers.

By investigating several pairs of Tr and Src amino acids, Chikaraishi et al. (2009) identified glutamic acid and phenylalanine as the best combination to return the most accurate estimation of the trophic position of consumers (TP Glu/Phe ). Since then, several studies further suggested that using the average δ 15 N values of Tr and Src amino acids of multiple amino acids may provide greater statistical power to TP calculations than a single pair of amino acids (e.g., Bradley et al., 2015;Décima, Landry, & Popp, 2013;Nielsen, Popp, & Winder, 2015;Sherwood et al., 2011).
The unique metabolic pathway of individual amino acids can affect their isotopic behavior (whether Tr or Scr amino acids). Within heterotrophs, these differential enrichment (or depletion) patterns determine the amount of intertrophic isotopic discrimination (e.g., Chikaraishi et al., 2007Chikaraishi et al., , 2009Ohkouchi, Ogawa, Chikaraishi, Tanaka, & Wada, 2015). For instance, it has been proposed that catabolic deamination (preceding transamination) of Tr amino acids causes the preferential cleavage of the 14 N amino group, resulting in an accumulation of 15 N (by up to ~3-8‰ per trophic level) in the Tr amino acids of consumer (Chikaraishi et al., 2007). In in vitro trials, Miura and Goto (2012) reported that the magnitude of isotopic discrimination of glutamic acid strongly correlates with its deamination flux (i.e., the deamination of a large pool generates greater isotopic discrimination compared to that from a smaller pool). However, the metabolism of Src amino acids does not involve the formation or cleaving of carbon-nitrogen bonds. Therefore, there is negligible isotopic discrimination in the Src amino acids between consumer and diet (Chikaraishi et al., 2007). The metabolic routing of amino acids may invoke alternative patterns of isotopic discrimination, particularly in the carbon isotopes of nonessential amino acids (McMahon, Fogel, Elsdon, & Thorrold, 2010). Although discrimination in nitrogen isotopes associated with metabolic routing has not been evidenced (Chikaraishi et al., 2007), the balance of amino acids, lipids, and carbohydrates as metabolic energy sources can potentially cause a significant variation in the isotopic discrimination of amino acids (Blanke et al., 2017;Chikaraishi, Steffan, Takano, & Ohkouchi, 2015;McMahon, Thorrold, Elsdon, & McCarthy, 2015).
With the exception of photosynthesis, there are several metabolic parallels between plants and heterotrophs ( Figure 1, cf: Buchanan, Gruissem, & Jones, 2000). For example, plants can store photosynthetically fixed energy in form of carbohydrates, lipids, and/or amino acids (Buchanan et al., 2000;Chapin, Schulze, & Mooney, 1990;Kermode, 2011;Millard, 1996). During periods of lean photosynthesis, such as heterotrophs, the catabolism of these storage compounds releases energy (i.e., ATP) that is subsequently used for the anabolism of new constituents (Buchanan et al., 2000), particularly in organs without chloroplasts (e.g., flower and root). If this catabolism within autotrophic biomass involves the deamination of amino acids, the resulting residual pool of amino acids (particularly for Tr amino acids) will be more enriched in 15 N than the original source pool. The mobilization and assimilation of these 15 N-enriched amino acids may generate isotopic differences between the source pool and newly synthesized biomass. Unlike "inter"-trophic isotopic discrimination that involves two separate organisms with unique trophic identities (e.g., Chikaraishi et al., 2007), "intra"-trophic isotopic discrimination arises as a result of  (Reich, Walters, Tjoelker, Vanderklein, & Buschena, 1998). The intratrophic isotopic discrimination in plants, primarily an outcome of amino acid deamination, is therefore hardly detectable during the growing season when metabolism is largely geared toward photosynthesis. The significant reduction or even absence of photosynthesis during winter dormancy, however, temporarily severs the energy supply for the homeostasis (Damesin, 2003). During this time, plants (mostly deciduous) must meet the energetic demands for the maintenance of basic cellular function through the catabolism of organic storage compounds (Arora, Wisniewski, & Scorza, 1992;Gomez & Faurobert, 2002;Loescher, McCamant, & Keller, 1990;Olofinboba, 1969), which may include deamination of storage amino acids, that ultimately results in the intratrophic isotopic discrimination within a plant tissue. Indeed, Takizawa and Chikaraishi (2014) first reported that sweet potato sprout grown in the absence of light has an unusually high TP Glu/Phe value of 2.2. Given that sprouting occurred in dark, and in absence of photosynthesis, sprout biomass likely recorded the 15 N-enrichment derived from the amino acid deamination during catabolism.
We hypothesize that plant organs lacking chloroplasts may undergo intratrophic isotopic discrimination via the aforementioned mechanisms. This leads to an increased δ 15 N Tr values in these organs, and therefore to an ecologically erroneous overestimation (TP Glu/ Phe > 1.0) for plant trophic position. The objective of our study was to investigate whether indeed there was a measurable amount of intratrophic isotopic discrimination between chloroplast-bearing leaves and chloroplast-lacking flowers, and to assess its implication for trophic position calculation.

| Leaf and flower samples
Flowers and mature leaves of eight deciduous trees and two annual plants were collected in their blooming season (February-May) from either a farm or a house garden in Yugawara, Japan (35°08′N, 139°07E) (Table S1). These plants commonly begin to grow leaves in spring and completely lose their leaves in autumn. They were classified into Type I and Type II, with respect to the timing of their bloom relative to leaf emergence ( Figure 2). Type I plants included Both Type I and Type II plants were chosen as they are commonly found in agricultural area and/or house gardens in the temperate region of Japan. Approximately ten leaves and ten flowers were collected for each plant. The collected samples were cleaned with distilled water to remove surface contaminants, homogenized to a fine powder using a Tube-Mill (IKA, Staufen, Germany), freeze-dried, and then stored at −20°C.

| Analysis of the δ 15 N AA values
The samples were prepared for the δ 15 N AA analysis after HCl hydrolysis and N-pivaloyl/isopropyl (Pv/iPr) derivatization, according to the procedure in Chikaraishi et al. (2009). In brief, the homogenized samples were hydrolyzed using 12 M HCl at 110°C overnight (>12 hr).

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| Calculation of the TP Glu/Phe values
The TP Glu/Phe value was calculated from the observed δ 15 N values of glutamic acid (δ 15 N Glu ) and phenylalanine (δ 15 N Phe ), using equation (1) with the β Glu/Phe and TDF Glu/Phe (inter-TDF Glu/Phe ) being −8.

| The δ 15 N AA and TP Glu/Phe values in leaves and flowers
Leaves and flowers fell within a similar but wide range in the δ 15 N AA value within Type I and Type II plants (Type I leaves = −2.5 ± 8.2‰; Type I flowers = −3.5 ± 5.9‰ and Type II leaves = −6.3 ± 9.2‰; Type II flowers = −5.9 ± 9.2‰; mean ± 1σ, Table 1). As expected, TP Glu/Phe value for both Type I and Type II leaves reported a mean of 1.0 ± 0.1 (Figure 3), consistent with previously reported values (1.0 ± 0.2) of plant samples such as leaves, nuts, and sap (Chikaraishi et al., 2011Steffan et al., 2013). However, the TP Glu/Phe value of Type I flowers (2.2 ± 0.2) was significantly higher than that of Type II flowers (1.0 ± 0.1) (t 8 = 10.63, p < .001). Additionally, the TP Glu/Phe value of Type I flowers was significantly higher than the functional trophic position of autotrophs (TP ~1.0) in any ecosystem (t 4 = 11.05, p < .001).
However, whether this well-documented trophic identity applies to all organs within an individual plant has not been fully investigated (Takizawa & Chikaraishi, 2014). Our results indicate that even within a single plant, the Δδ 15 N Glu−Phe value (and therefore the TP Glu/Phe value) of different organs can vary significantly (Figures 3 and 4). We propose possible physiological scenarios that could contribute to this isotopic heterogeneity between leaves and flowers of Type I and Type II plants, potentially skewing their trophic identities. have shown that antifreeze protein helps deciduous trees to survive through winter dormancy (Arora et al., 1992;Hon, Griffith, Mlynarz, Kwok, & Yang, 1995). Because these proteins are not required during spring, they may be subsequently deaminated (Arora et al., 1992). The residual pool of 15 N-enriched amino acids generates "intra"-trophic discrimination, especially in Tr amino acids (e.g., glutamic acid), and when incorporated in newly synthesized Type I floral tissue, inflates their TP Glu/Phe value ( Figure 5). F I G U R E 5 Possible metabolic states for flowering of the Type I plants, which includes deamination of amino acids and therefore alternative isotopic discrimination leading to significant elevation in the TP Glu/Phe value of amino acids in flowers Type II plants represent a different phenology where bloom occurs in the presence of actively photosynthesizing leaves. As energy fixed by active photosynthesis is sufficient to support bloom, this may preclude the necessity of deamination of amino acids ( Figure 5), which can explain the negligible intra-trophic isotopic discrimination of amino acids in Type II plants. Therefore, the TP Glu /Phe value of Type   II leaves and Type II flowers remains comparable with each other, and to the expected value of 1.0.

| Intratrophic isotopic discrimination
We suggest the following equation (2)

| The δ 15 N values of phenylalanine in leaves and flowers
There was a large variation in the δ 15 N value of phenylalanine within However, this variation is consistent with data published in previous studies using leaves collected from the same farm (10.6 ± 3.8‰) (Chikaraishi et al., 2011.

| Implications
CSIA has expanded the ecologists' toolbox by allowing high-resolution insights into trophic interactions. However, little information is available about the factors controlling inter-and/or intratrophic isotopic discrimination of amino acids in plants, animals, fungi, and bacteria Gutiérrez-Rodríguez, Décima, Popp, & Landry, 2014;McMahon et al., 2015;Steffan, Chikaraishi, Currie, et al., 2015). Our findings reveal unique isotopic heterogeneity among wild plant organs, which can confound trophic estimations of these plants and the consumers that they support. For example, will feeding (2) TDF � Glu/Phe = δ 15 N Organ,Glu −δ 15 N Organ,Phe −β on Type I pollen in early spring elevate the TP Glu/Phe of pollinators and nectarivores above that expected of herbivores (>2.0)? How will the preferential feeding of herbivores on early spring flowers or new leaves imprint on the trophic positions of higher order consumers?
What will be the trophic identity of the detritus derived from Type I flowers as they become a basal resource in the brown food web?
Ideally, the TP Glu/Phe (or TP Tr/Src ) value enables isotopic ecologists to deduce the ecological function (e.g., primary producer, herbivore, omnivore, and carnivore) of organisms (Bradley et al., 2015;Chikaraishi et al., 2014;Nielsen et al., 2015;Steffan, Chikaraishi, Horton, et al., 2015). However, our data indicate that the TP Glu/Phe value does not always reflect an organism's functional trophic position in the food web. For instance, although Type I flowers returned a TP Glu/Phe value of 2.2, such value is typical of omnivores (TP > 2.0) and certainly does not represent the "functional" trophic identity of plants and their organs.
Therefore, it appears that during sum of intra-and intertrophic isotopic discriminations, organismal TP Glu/Phe values may represent "energetic" tendencies rather than the organism's true functional trophic position in food webs. These differences between the energetic and functional trophic positions arising from intratrophic isotopic discrimination can complicate food web studies. We therefore encourage continued investigations to reevaluate how CSIA-derived trophic position correlates with the δ 15 N AA values of organisms in food webs.