BACKGROUND
plant ecologists increasingly recognize that plants can capture nitrogen in a variety of different chemical forms, ranging from inorganic forms such as nitrate and ammonium to as many as  <dig> different amino acids  <cit> .
this access to organic pools of nitrogen has been argued to increase plant access to nitrogen, especially in nitrogen limited habitats e.g.
<cit> .
additionally, ecologists have argued that plant access to a variety of different forms of nitrogen may also influence population and community processes by allowing increased dimensions of niche partitioning mediated through differences in nitrogen preference  <cit> .
however, while ecologists have obtained large amounts of data investigating whether plants can or cannot capture specific amino acids, there is surprisingly little data that links this uptake to plant growth and performance.
we argue that it is not enough to simply show that plants will use certain forms of nitrogen, but for ideas surrounding potential ecological dynamics we need data that show these different types of nitrogen are actually of value to plants.
generally, uptake rates for amino acids are considerably lower compared to inorganic forms of nitrogen  <cit> , though they may be higher  <cit> .
data suggests that, when grown with amino acids as the sole nitrogen source, the model plant arabidopsis thaliana  heynh, var.
columbia) was only capable of achieving between 1% and 50% of its potential vegetative growth on an identical concentration of nitrate  <cit> .
in general it appears that most of the amino acids that plants can use as a source of nitrogen depress plant growth relative to mineral sources of nitrogen  <cit>  while only a minority of amino acids can occasionally increase plant growth  <cit>  or at least match plant growth on mineral forms of nitrogen  <cit> .
however, almost all the available data come from single nitrogen nutrition studies, and multiple nitrogen types in mixture are rarely offered to plants.
yet, plants in ecological communities will experience soil with multiple nitrogen types available simultaneously, and it is important to understand the nature of any interactions among nitrogen types.
this means that plants may have to select among nitrogen types, and that there may also be positive or negative interactions among nitrogen types in their contribution to plant performance.
in this paper, three experiments are presented which were designed to explore the consequences of single and mixed nitrogen nutrition for the growth and potential reproductive output of the model plant arabidopsis thaliana  heynh, var.
columbia).
the goal was to explore links between mixed nitrogen nutrition and plant growth and reproductive output.
in experiment  <dig>  correlations between traits measured during the vegetative growth phase and lifetime reproductive effort at senescence were examined.
here, the goal was to be able to estimate potential lifetime performance of individuals harvested prior to senescence.
in experiment  <dig>  plants were offered one of three types of nitrogen at nine concentrations and plant traits were measured to understand how single nitrogen nutrition influenced plant growth and reproductive output.
this was done as a control, to examine plant performance on single nitrogen nutrition.
in experiment  <dig>  mixed nitrogen nutrition was examined by growing plants with two types of nitrogen simultaneously at  <dig> different ratios for all pairwise combinations of nitrate, glutamine and asparagine.
the relative amounts of each form of nitrogen captured, and plant traits were each measured to determine how plant growth and reproductive effort varied on mixed nitrogen nutrition.
it was hypothesized that plants would be capable of growing and reproducing when fed amino acids, but those amino acids would contribute relatively little to plant growth and reproduction compared to mineral sources of nitrate.
it was also hypothesized that given two types of nitrogen; plants would be unlikely to preferentially capture amino acids.
at the end of the manuscript, we return to ideas about ecology and evolution and suggest a closer link between questions asked by plant ecologists working at all levels of organization would greatly advance the general understanding of amino acid nutrition in plant systems.
RESULTS
experiment 1: traits and reproductive effort
in experiment  <dig>  plants were grown at a range of different nutrient availabilities to generate variability in size, and values of plant traits in the middle of their growth cycle  were linked to lifetime seed production at senescence  through regression.
we measured leaf number, rosette diameter, flower number and scored plant stress based on leaf colour.
leaf number , rosette diameter  and flower number  at 4 weeks of age were all positively correlated with lifetime seed production .
physiological stress  was negatively correlated with lifetime seed production indicating that high nutrient stress was generally related to low reproductive output .
though all traits were significantly correlated with lifetime seed production, rosette diameter at 4 weeks of age was the most efficient predictor of lifetime seed production at senescence .
this analysis allows us to link vegetative growth traits to potential reproductive output in plants.
experiment 2: single nitrogen nutrition
in this experiment plants were grown with only one type of nitrogen  at nine different concentrations  to investigate the potential for each nitrogen type alone to contribute to plant growth and reproductive output.
data indicate that plants were able to grow and produce flowers on all nitrogen types confirming that nitrate, glutamine and asparagine were each viable sole sources of nitrogen for a. thaliana .
however, the three types of nitrogen had significantly different effects on plant performance ; in general, plants performed best by any metric on nitrate, intermediate on glutamine and poorest on asparagine .
although plants could grow and reproduce with asparagine as the sole source of nitrogen , the plants were often small , had few flowers  and were obviously nutrient stressed .
for any of the three nitrogen types, the concentration of nitrogen was positively correlated with flower number and rosette diameter .
within the narrow range of nitrogen concentrations used , nutrient stress and leaf number were relatively invariant regardless of nitrogen concentration .
however, there was a marginally significant trend towards decreasing stress with increasing nitrate concentration compared to relatively constant stress for plants supplied either form of amino acid .
the significant interaction between nitrogen type and concentration for flower number can be explained because glutamine and asparagine produced similarly low numbers of flowers at low concentrations causing the regression lines to cross at low concentrations .
experiment 3: mixed nitrogen nutrition
in this experiment plants were supplied with two types of nitrogen simultaneously, at nine different ratios  such that the total amount of nitrogen was always 1 mm.
the goal was to investigate whether plants had any preferential uptake when offered two types of nitrogen at the same time, and to examine how mixed nitrogen nutrition influenced plant traits and reproductive effort relative to single nitrogen nutrition.
we asked three questions:  did the plants capture amino acids intact?
did the plants show any preferential uptake when offered two nitrogen types simultaneously?
how did plant traits vary based on the ratios of nitrogen available?
first, the data from dual labeled amino acids suggest amino acids were captured intact.
the %15 n and %13c enrichment of each plant were compared and indeed, %15 n and %13c enrichment were strongly positively correlated for both glutamine uptake  and asparagine uptake  .
second, to compare the relative amounts of each nitrogen type captured, the known %15 n supplied in the growth media was compared to the measured %15 n in each plant using linear regressions .
the key test here was whether the slope of available versus captured differed from  <dig>  and whether it was greater than, or less than,  <dig>  in each case, the slope of the observed relationship was significantly less than  <dig>  for nitrate and glutamine , nitrate and asparagine  and glutamine and asparagine .
this indicated that the plants preferentially captured the unlabeled nitrogen source in each treatment.
nitrate was preferred in the nitrate + glutamine treatment , and in the nitrate + asparagine treatment , and glutamine was preferred in the glutamine + asparagine treatment .
from this it was concluded that plant preferences followed a consistent hierarchy of nitrate > glutamine > asparagine across all pairs of nitrogen .
this matched the predicted preferences from the single nitrogen data in experiment  <dig> .
third, the influence of nitrogen mixtures and ratios on plant growth and performance was investigated.
in this experiment there was always 1 mm of total nitrogen and only the ratio of nitrogen types offered was manipulated.
as a result there was more overlap of plant traits among treatments which resulted in significant interactions among nitrogen types and ratio for most plant traits .
significant interactions were recorded for flower number, rosette diameter and leaf number .
these interactions were interpreted as stemming from the fact that plants performed similarly when nitrate was present regardless of which amino acid co-occurred with nitrate causing overlap of the regression lines between the nitrate + glutamine and nitrate + asparagine treatments .
it was also observed that, as the ratios available shifted in favour of the preferred nitrogen type, plant performance generally increased by all metrics, but the slopes were relatively shallow demonstrating that this increase was marginal .
discussion
increasingly, plant ecologists working at all levels of organization have become interested in the role of amino acid nutrition in the lives of plants  <cit> .
many authors report uptake of amino acids, but this uptake is only rarely linked to plant biomass production or potential reproductive effort of plants.
our data suggest that amino acids are relatively poor sources of nitrogen compared to nitrate, and that amino acids contribute a very small amount to growth and potential reproductive output in a. thaliana .
though a. thaliana plants in this study captured amino acids intact  and could grow and reproduce on both asparagine and glutamine as the sole nitrogen source , plants grown on amino acids were of often less than half the size of plants grown with nitrate as the only nitrogen source .
mixed nitrogen nutrition produced similar results, with the plants grown only on asparagine  + glutamine achieving only a fraction of the growth of plants supplied with nitrate and either amino acid .
plants also appear to preferentially capture nitrogen types following a consistent hierarchy of preferences, with nitrate > glutamine > asparagine .
not only did plants grow poorly on amino acids, but the mixed nitrogen nutrition experiment revealed that the contribution of each nitrogen form was not additive.
instead, there appeared to be a negative interaction between different nitrogen types that was costly to the plant in terms of potential performance.
for example, plants grown solely on nitrate in experiment  <dig> achieved the highest performance in any of our experiments, with a mean of  <dig>  flowers and a mean rosette diameter of  <dig>  mm at the highest concentration  of nitrate .
however, in experiment  <dig>  with an identical concentration of nitrate plus additional nitrogen in the form of amino acids , plants achieved a mean of  <dig>  flowers and a mean rosette diameter of only  <dig>  mm.
that is, even though the amount of nitrate was identical, and the total amount of nitrogen was actually higher, plants had only 29% as many flowers, and achieved only 75% of the vegetative growth with mixed nitrogen nutrition  compared to pure nitrate nutrition .
this lower performance in the presence of similar nitrogen concentrations suggests that amino acid capture comes at some net-cost to growth and reproduction which outstripped any benefits that might be expected from increased access to nitrogen.
these differences were not due to differences in calcium, as all three solutions contained an identical concentration of ca <dig>  the differences among these treatments might have been due to differences in ph between solutions , however with a difference of ± <dig> , this ph range was within the margin of error of many experiments that attempt to control ph.
plants grew significantly worse on asparagine compared to glutamine .
this might have been due to differences in uptake kinetics.
these uptake kinetics are well described for the model plant a. thaliana.
at concentrations similar to the range used in the present experiment, a. thaliana has almost identical uptake rates for both glutamine and asparagine .
despite the nearly equal maximum influx rates, and the fact that both amino acids contain two nitrogen atoms, the results presented here show that these two amino acids are not equal in terms of their contribution to plant growth and reproduction .
in all experiments vegetative traits related to potential reproductive output were significantly lower when plants were grown with asparagine compared to glutamine.
this suggests that uptake of asparagine may result in some unknown fitness cost compared to glutamine in terms of uptake, transport and/or metabolism.
additionally, even though asparagine and glutamine have potentially equal maximum influx rates when offered singly  <cit> , when we offered the two amino acids in mixture, the plants in this study preferentially captured glutamine over asparagine.
since influx rates and affinities are similar for glutamine and asparagine  <cit> , we expected a. thaliana to capture these two amino acids in identical amounts.
however, this was not what we observed; instead the plants somehow preferentially captured glutamine over asparagine .
this preferential uptake suggests that influx rates are not constant and plants may modulate their nitrogen uptake when provided with multiple types of nitrogen .
for example, tomato , is capable of modulating the expression of genes related to nitrogen uptake such that uptake sites are only expressed for those nitrogen types that are available  <cit> .
these genes in a. thaliana and tomato are homologous  <cit> , supporting the idea that nutrient uptake in is neither passive, nor constant.
however, most authors seem to assume that uptake is both passive and constant, even though data that might test this hypothesis are extremely limited.
if plants do modulate their nitrogen uptake it may be part of a plant’s overall nitrogen foraging strategy in the same way that animals may choose prey that maximize benefits and minimize costs, plants may choose nitrogen types adaptively  <cit> .
the standard ecological hypothesis for why plants capture amino acids, is that it enhances access to nitrogen, and presumably is a net-benefit to plants  <cit> .
however, given that plants seem to grow poorly on amino acids and our results revealed that amino acids negatively interact with mineral sources to further reduce plant growth and reproductive potential this led us to ask: why does a. thaliana bother to capture amino acids?
there is clear evidence that plants do capture amino acids in field conditions  <cit> , and many genes for uptake and transport of amino acids have been identified  <cit> , indicating the potential for selection to act on the allelic variability in these genes associated with uptake.
one hypothesis is that plant uptake is constant and hardwired, and plants simply have no “choice” in the matter.
plants simply capture whatever nitrogen sources are nearby in accordance with passive michaelis-menten kinetics.
though this seems to be prevailing wisdom, we reject this hypothesis, as evolutionarily unstable: any genes that do not offer a net benefit to plants will be removed from the population via natural selection, thus there must be some benefit to amino acid uptake that was hidden in our simple experiment.
a second hypothesis is that there may be a pleiotropic effect where genes associated with nitrogen uptake are also linked with other phenotypic traits that are under stronger selection and thus sub-optimal uptake is maintained because of selection on other traits.
there are several knock-out mutants for genes associated with nitrate uptake  <cit>  and amino acid uptake  <cit>  that might aid in the study of this question.
if amino acid uptake genes are advantageous because of pleiotropy, we would expect a plant with genes associated with amino acid uptake to perform poorly compared to wild type plants even under pure nitrate nutrition.
finally, a third hypothesis is that, as in many aspects of plant nutrient foraging, the answer may be in competitive interactions among plants e.g.
<cit> .
specifically, that amino acid nutrition might not necessarily be an adaptation to enhance nitrogen uptake per se, but rather it might be an adaptation to enhance nitrogen uptake relative to potential competitors through resource partitioning.
evolutionary game theory has revealed that many aspects of plant-plant interactions produce evolutionary arms races that result in a tragedy of the commons  <cit> , and this might be what happened in our experiment.
that is, past competition for nitrogen might have produced character displacement causing plants to shift to less desirable nitrogen sources as a result of competition  <cit> .
if this is true, the benefits of mixed nitrogen nutrition might only be apparent when plants face competition for nitrogen  <cit> .
for example, plants preferentially place roots in nutrient rich soil patches, but the benefits of this behaviour only become apparent when plants compete for patches  <cit> .
when plants grow alone, this root foraging behaviour often has no obvious benefits  <cit> , and may even be costly  <cit> .
this occurs because plants over-proliferate roots  in order to pre-empt competitor resource supply, but when competitors are absent this over-proliferation is not advantageous.
thus, one explanation for our results is that something similar occurs for amino acid uptake, where plants are attempting to partition resources with their competitors, but in the absence of competition this is not advantageous.
however, there is currently limited data to examine this question.
based on our results, and the lack of information about how uptake is altered by the presence of a competitor, two paths forward are suggested.
first, more data are needed from ecologists about availability and importance of amino acids from a range of species and ecosystems.
much of the data surrounding uptake of amino acids comes from model plants and crop species.
while we can certainly learn what is possible from model plants, studies of model plants do not provide information on the range and diversity of responses that may occur among plants in general.
in terms of ecosystems, much of the data that suggests an important role of amino acids in shaping competitive and community outcomes among plants comes from relatively cool boreal, arctic and alpine systems where organic nitrogen is the dominant form in the soil  <cit> .
however, the link between nitrogen preference and community composition has been much more elusive in more temperate systems, where decomposition of organic nitrogen occurs at a higher rate  <cit>  and is rarely studied in tropical systems.
thus, while it is intriguing to think that amino acid uptake might form a major component of the ecology and evolution of plants e.g.
<cit> , these data from arctic and alpine systems linking amino acids preferences to plant community composition might be the exception rather than the rule.
the “acid test” for the generality of these ideas surrounding nitrogen partitioning for the ecology and evolution of plants worldwide will come from tropical and temperate systems where decomposition rates lead to higher turnover of organic pools, and higher microbial activity leads to more intense competition with microbes  <cit> .
second, more data are needed from plant ecophysiologists that examine the role of amino acid nutrition when plants compete and when they are offered mixed nitrogen sources.
key questions that have little data in the literature are: do patterns of uptake change as a function of the presence or absence of competitors?
and do patterns of uptake change in mixed versus single nitrogen solutions?
almost all experiments in the literature are based upon uptake of a single nitrogen type by plants that do not experience competition.
however, there are a small number of studies that shed light on this process.
for example, miller et al.
<cit>  used neighbour removals and 15 n tracers in the field to examine how neighbour identity influenced capture of ammonium, nitrate or glycine.
they found a strong influence of neighbour identity in shaping the types and amounts of nitrogen captured  <cit> .
this pattern might be caused purely by patterns of resource depletion by neighbours e.g.
<cit> , but it might also be caused by plants adjusting their uptake kinetics in the presence of neighbours.
in another study abbes et al.
<cit>  provided onion plants  with nitrate and ammonium at a variety of ratios using a design that was similar to our experiment  <dig>  they found that depletion trajectories of both nitrate and ammonium differed substantially based on the ratios of nitrogen available in the environment  <cit> .
this supports the idea that uptake kinetics are labile and may be adjusted based on availability of nitrogen types in the environment.
however, remarkably few data are available, and most studies do not provide multiple nitrogen types, adjust ratios, or examine the effects of competition.
given the few interesting results that are available  <cit> , additional studies that track uptake kinetics and gene expression in mixed nitrogen nutrition experiments, and when plants compete would provide key information about the plasticity plants possess in uptake kinetics, and greatly advance the general understanding of plant nitrogen nutrition in all fields of plant sciences.
CONCLUSIONS
it is now recognized that plants regularly capture amino acids along with mineral sources of nitrogen, and it is generally assumed that amino acid nutrition enhances access to nitrogen and is a net fitness benefit to plants  <cit> .
the results presented above suggest that at concentrations that are similar to those found in natural soils, amino acids contribute relatively little to potential vegetative growth or reproductive output in a. thaliana relative to inorganic forms of nitrogen such as nitrate.
in all experiments plants achieved lower performance when supplied with amino acids, and preferentially captured nitrate over amino acids when given nitrogen in mixture.
furthermore, there was a negative interaction between amino acids and nitrate when plants were fed with mixed nitrogen nutrition.
these plants had lower overall growth and potential reproductive output then plants fed a similar amount of a single type of nitrogen.
this suggests some cost associated with and expanded nitrogen diet, and raises questions about why plants bother to capture amino acids from soil.
faced with these curious results, we hypothesize that the importance of amino acid nutrition may be related to nitrogen competition, and this role may only be obvious in experiments that compare plants grown alone to those grown with neighbours.
regardless of the outcome, examining this untested hypothesis will provide valuable data about whether and how plants adjust their nutrient uptake kinetics.

 plant ecologists increasingly recognize that plants can capture nitrogen in a variety of different chemical forms, ranging from inorganic forms such as nitrate and ammonium to as many as different amino acids. this access to organic pools of nitrogen has been argued to increase plant access to nitrogen, especially in nitrogen limited habitats e.g.. additionally, ecologists have argued that plant access to a variety of different forms of nitrogen may also influence population and community processes by allowing increased dimensions of niche partitioning mediated through differences in nitrogen preference. however, while ecologists have obtained large amounts of data investigating whether plants can or cannot capture specific amino acids, there is surprisingly little data that links this uptake to plant growth and performance. we argue that it is not enough to simply show that plants will use certain forms of nitrogen, but for ideas surrounding potential ecological dynamics we need data that show these different types of nitrogen are actually of value to plants. generally, uptake rates for amino acids are considerably lower compared to inorganic forms of nitrogen, though they may be higher. data suggests that, when grown with amino acids as the sole nitrogen source, the model plant arabidopsis thaliana heynh, var. columbia) was only capable of achieving between 1% and 50% of its potential vegetative growth on an identical concentration of nitrate. in general it appears that most of the amino acids that plants can use as a source of nitrogen depress plant growth relative to mineral sources of nitrogen while only a minority of amino acids can occasionally increase plant growth or at least match plant growth on mineral forms of nitrogen. however, almost all the available data come from single nitrogen nutrition studies, and multiple nitrogen types in mixture are rarely offered to plants. yet, plants in ecological communities will experience soil with multiple nitrogen types available simultaneously, and it is important to understand the nature of any interactions among nitrogen types. this means that plants may have to select among nitrogen types, and that there may also be positive or negative interactions among nitrogen types in their contribution to plant performance. in this paper, three experiments are presented which were designed to explore the consequences of single and mixed nitrogen nutrition for the growth and potential reproductive output of the model plant arabidopsis thaliana heynh, var. columbia). the goal was to explore links between mixed nitrogen nutrition and plant growth and reproductive output. in experiment correlations between traits measured during the vegetative growth phase and lifetime reproductive effort at senescence were examined. here, the goal was to be able to estimate potential lifetime performance of individuals harvested prior to senescence. in experiment plants were offered one of three types of nitrogen at nine concentrations and plant traits were measured to understand how single nitrogen nutrition influenced plant growth and reproductive output. this was done as a control, to examine plant performance on single nitrogen nutrition. in experiment mixed nitrogen nutrition was examined by growing plants with two types of nitrogen simultaneously at different ratios for all pairwise combinations of nitrate, glutamine and asparagine. the relative amounts of each form of nitrogen captured, and plant traits were each measured to determine how plant growth and reproductive effort varied on mixed nitrogen nutrition. it was hypothesized that plants would be capable of growing and reproducing when fed amino acids, but those amino acids would contribute relatively little to plant growth and reproduction compared to mineral sources of nitrate. it was also hypothesized that given two types of nitrogen; plants would be unlikely to preferentially capture amino acids. at the end of the manuscript, we return to ideas about ecology and evolution and suggest a closer link between questions asked by plant ecologists working at all levels of organization would greatly advance the general understanding of amino acid nutrition in plant systems.  experiment 1: traits and reproductive effort in experiment plants were grown at a range of different nutrient availabilities to generate variability in size, and values of plant traits in the middle of their growth cycle were linked to lifetime seed production at senescence through regression. we measured leaf number, rosette diameter, flower number and scored plant stress based on leaf colour. leaf number, rosette diameter and flower number at 4 weeks of age were all positively correlated with lifetime seed production. physiological stress was negatively correlated with lifetime seed production indicating that high nutrient stress was generally related to low reproductive output. though all traits were significantly correlated with lifetime seed production, rosette diameter at 4 weeks of age was the most efficient predictor of lifetime seed production at senescence. this analysis allows us to link vegetative growth traits to potential reproductive output in plants. experiment 2: single nitrogen nutrition in this experiment plants were grown with only one type of nitrogen at nine different concentrations to investigate the potential for each nitrogen type alone to contribute to plant growth and reproductive output. data indicate that plants were able to grow and produce flowers on all nitrogen types confirming that nitrate, glutamine and asparagine were each viable sole sources of nitrogen for a. thaliana. however, the three types of nitrogen had significantly different effects on plant performance; in general, plants performed best by any metric on nitrate, intermediate on glutamine and poorest on asparagine. although plants could grow and reproduce with asparagine as the sole source of nitrogen, the plants were often small, had few flowers and were obviously nutrient stressed. for any of the three nitrogen types, the concentration of nitrogen was positively correlated with flower number and rosette diameter. within the narrow range of nitrogen concentrations used, nutrient stress and leaf number were relatively invariant regardless of nitrogen concentration. however, there was a marginally significant trend towards decreasing stress with increasing nitrate concentration compared to relatively constant stress for plants supplied either form of amino acid. the significant interaction between nitrogen type and concentration for flower number can be explained because glutamine and asparagine produced similarly low numbers of flowers at low concentrations causing the regression lines to cross at low concentrations. experiment 3: mixed nitrogen nutrition in this experiment plants were supplied with two types of nitrogen simultaneously, at nine different ratios such that the total amount of nitrogen was always 1 mm. the goal was to investigate whether plants had any preferential uptake when offered two types of nitrogen at the same time, and to examine how mixed nitrogen nutrition influenced plant traits and reproductive effort relative to single nitrogen nutrition. we asked three questions: did the plants capture amino acids intact? did the plants show any preferential uptake when offered two nitrogen types simultaneously? how did plant traits vary based on the ratios of nitrogen available? first, the data from dual labeled amino acids suggest amino acids were captured intact. the %15 n and %13c enrichment of each plant were compared and indeed, %15 n and %13c enrichment were strongly positively correlated for both glutamine uptake and asparagine uptake. second, to compare the relative amounts of each nitrogen type captured, the known %15 n supplied in the growth media was compared to the measured %15 n in each plant using linear regressions. the key test here was whether the slope of available versus captured differed from and whether it was greater than, or less than, in each case, the slope of the observed relationship was significantly less than for nitrate and glutamine, nitrate and asparagine and glutamine and asparagine. this indicated that the plants preferentially captured the unlabeled nitrogen source in each treatment. nitrate was preferred in the nitrate + glutamine treatment, and in the nitrate + asparagine treatment, and glutamine was preferred in the glutamine + asparagine treatment. from this it was concluded that plant preferences followed a consistent hierarchy of nitrate > glutamine > asparagine across all pairs of nitrogen. this matched the predicted preferences from the single nitrogen data in experiment. third, the influence of nitrogen mixtures and ratios on plant growth and performance was investigated. in this experiment there was always 1 mm of total nitrogen and only the ratio of nitrogen types offered was manipulated. as a result there was more overlap of plant traits among treatments which resulted in significant interactions among nitrogen types and ratio for most plant traits. significant interactions were recorded for flower number, rosette diameter and leaf number. these interactions were interpreted as stemming from the fact that plants performed similarly when nitrate was present regardless of which amino acid co-occurred with nitrate causing overlap of the regression lines between the nitrate + glutamine and nitrate + asparagine treatments. it was also observed that, as the ratios available shifted in favour of the preferred nitrogen type, plant performance generally increased by all metrics, but the slopes were relatively shallow demonstrating that this increase was marginal. discussion increasingly, plant ecologists working at all levels of organization have become interested in the role of amino acid nutrition in the lives of plants. many authors report uptake of amino acids, but this uptake is only rarely linked to plant biomass production or potential reproductive effort of plants. our data suggest that amino acids are relatively poor sources of nitrogen compared to nitrate, and that amino acids contribute a very small amount to growth and potential reproductive output in a. thaliana. though a. thaliana plants in this study captured amino acids intact and could grow and reproduce on both asparagine and glutamine as the sole nitrogen source, plants grown on amino acids were of often less than half the size of plants grown with nitrate as the only nitrogen source. mixed nitrogen nutrition produced similar results, with the plants grown only on asparagine  + glutamine achieving only a fraction of the growth of plants supplied with nitrate and either amino acid. plants also appear to preferentially capture nitrogen types following a consistent hierarchy of preferences, with nitrate > glutamine > asparagine. not only did plants grow poorly on amino acids, but the mixed nitrogen nutrition experiment revealed that the contribution of each nitrogen form was not additive. instead, there appeared to be a negative interaction between different nitrogen types that was costly to the plant in terms of potential performance. for example, plants grown solely on nitrate in experiment achieved the highest performance in any of our experiments, with a mean of flowers and a mean rosette diameter of  mm at the highest concentration of nitrate. however, in experiment with an identical concentration of nitrate plus additional nitrogen in the form of amino acids, plants achieved a mean of flowers and a mean rosette diameter of only  mm. that is, even though the amount of nitrate was identical, and the total amount of nitrogen was actually higher, plants had only 29% as many flowers, and achieved only 75% of the vegetative growth with mixed nitrogen nutrition compared to pure nitrate nutrition. this lower performance in the presence of similar nitrogen concentrations suggests that amino acid capture comes at some net-cost to growth and reproduction which outstripped any benefits that might be expected from increased access to nitrogen. these differences were not due to differences in calcium, as all three solutions contained an identical concentration of ca the differences among these treatments might have been due to differences in ph between solutions, however with a difference of ±, this ph range was within the margin of error of many experiments that attempt to control ph. plants grew significantly worse on asparagine compared to glutamine. this might have been due to differences in uptake kinetics. these uptake kinetics are well described for the model plant a. thaliana. at concentrations similar to the range used in the present experiment, a. thaliana has almost identical uptake rates for both glutamine and asparagine. despite the nearly equal maximum influx rates, and the fact that both amino acids contain two nitrogen atoms, the results presented here show that these two amino acids are not equal in terms of their contribution to plant growth and reproduction. in all experiments vegetative traits related to potential reproductive output were significantly lower when plants were grown with asparagine compared to glutamine. this suggests that uptake of asparagine may result in some unknown fitness cost compared to glutamine in terms of uptake, transport and/or metabolism. additionally, even though asparagine and glutamine have potentially equal maximum influx rates when offered singly, when we offered the two amino acids in mixture, the plants in this study preferentially captured glutamine over asparagine. since influx rates and affinities are similar for glutamine and asparagine, we expected a. thaliana to capture these two amino acids in identical amounts. however, this was not what we observed; instead the plants somehow preferentially captured glutamine over asparagine. this preferential uptake suggests that influx rates are not constant and plants may modulate their nitrogen uptake when provided with multiple types of nitrogen. for example, tomato, is capable of modulating the expression of genes related to nitrogen uptake such that uptake sites are only expressed for those nitrogen types that are available. these genes in a. thaliana and tomato are homologous, supporting the idea that nutrient uptake in is neither passive, nor constant. however, most authors seem to assume that uptake is both passive and constant, even though data that might test this hypothesis are extremely limited. if plants do modulate their nitrogen uptake it may be part of a plant’s overall nitrogen foraging strategy in the same way that animals may choose prey that maximize benefits and minimize costs, plants may choose nitrogen types adaptively. the standard ecological hypothesis for why plants capture amino acids, is that it enhances access to nitrogen, and presumably is a net-benefit to plants. however, given that plants seem to grow poorly on amino acids and our results revealed that amino acids negatively interact with mineral sources to further reduce plant growth and reproductive potential this led us to ask: why does a. thaliana bother to capture amino acids? there is clear evidence that plants do capture amino acids in field conditions, and many genes for uptake and transport of amino acids have been identified, indicating the potential for selection to act on the allelic variability in these genes associated with uptake. one hypothesis is that plant uptake is constant and hardwired, and plants simply have no “choice” in the matter. plants simply capture whatever nitrogen sources are nearby in accordance with passive michaelis-menten kinetics. though this seems to be prevailing wisdom, we reject this hypothesis, as evolutionarily unstable: any genes that do not offer a net benefit to plants will be removed from the population via natural selection, thus there must be some benefit to amino acid uptake that was hidden in our simple experiment. a second hypothesis is that there may be a pleiotropic effect where genes associated with nitrogen uptake are also linked with other phenotypic traits that are under stronger selection and thus sub-optimal uptake is maintained because of selection on other traits. there are several knock-out mutants for genes associated with nitrate uptake and amino acid uptake that might aid in the study of this question. if amino acid uptake genes are advantageous because of pleiotropy, we would expect a plant with genes associated with amino acid uptake to perform poorly compared to wild type plants even under pure nitrate nutrition. finally, a third hypothesis is that, as in many aspects of plant nutrient foraging, the answer may be in competitive interactions among plants e.g.. specifically, that amino acid nutrition might not necessarily be an adaptation to enhance nitrogen uptake per se, but rather it might be an adaptation to enhance nitrogen uptake relative to potential competitors through resource partitioning. evolutionary game theory has revealed that many aspects of plant-plant interactions produce evolutionary arms races that result in a tragedy of the commons, and this might be what happened in our experiment. that is, past competition for nitrogen might have produced character displacement causing plants to shift to less desirable nitrogen sources as a result of competition. if this is true, the benefits of mixed nitrogen nutrition might only be apparent when plants face competition for nitrogen. for example, plants preferentially place roots in nutrient rich soil patches, but the benefits of this behaviour only become apparent when plants compete for patches. when plants grow alone, this root foraging behaviour often has no obvious benefits, and may even be costly. this occurs because plants over-proliferate roots in order to pre-empt competitor resource supply, but when competitors are absent this over-proliferation is not advantageous. thus, one explanation for our results is that something similar occurs for amino acid uptake, where plants are attempting to partition resources with their competitors, but in the absence of competition this is not advantageous. however, there is currently limited data to examine this question. based on our results, and the lack of information about how uptake is altered by the presence of a competitor, two paths forward are suggested. first, more data are needed from ecologists about availability and importance of amino acids from a range of species and ecosystems. much of the data surrounding uptake of amino acids comes from model plants and crop species. while we can certainly learn what is possible from model plants, studies of model plants do not provide information on the range and diversity of responses that may occur among plants in general. in terms of ecosystems, much of the data that suggests an important role of amino acids in shaping competitive and community outcomes among plants comes from relatively cool boreal, arctic and alpine systems where organic nitrogen is the dominant form in the soil. however, the link between nitrogen preference and community composition has been much more elusive in more temperate systems, where decomposition of organic nitrogen occurs at a higher rate and is rarely studied in tropical systems. thus, while it is intriguing to think that amino acid uptake might form a major component of the ecology and evolution of plants e.g., these data from arctic and alpine systems linking amino acids preferences to plant community composition might be the exception rather than the rule. the “acid test” for the generality of these ideas surrounding nitrogen partitioning for the ecology and evolution of plants worldwide will come from tropical and temperate systems where decomposition rates lead to higher turnover of organic pools, and higher microbial activity leads to more intense competition with microbes. second, more data are needed from plant ecophysiologists that examine the role of amino acid nutrition when plants compete and when they are offered mixed nitrogen sources. key questions that have little data in the literature are: do patterns of uptake change as a function of the presence or absence of competitors? and do patterns of uptake change in mixed versus single nitrogen solutions? almost all experiments in the literature are based upon uptake of a single nitrogen type by plants that do not experience competition. however, there are a small number of studies that shed light on this process. for example, miller et al. used neighbour removals and 15 n tracers in the field to examine how neighbour identity influenced capture of ammonium, nitrate or glycine. they found a strong influence of neighbour identity in shaping the types and amounts of nitrogen captured. this pattern might be caused purely by patterns of resource depletion by neighbours e.g., but it might also be caused by plants adjusting their uptake kinetics in the presence of neighbours. in another study abbes et al. provided onion plants with nitrate and ammonium at a variety of ratios using a design that was similar to our experiment they found that depletion trajectories of both nitrate and ammonium differed substantially based on the ratios of nitrogen available in the environment. this supports the idea that uptake kinetics are labile and may be adjusted based on availability of nitrogen types in the environment. however, remarkably few data are available, and most studies do not provide multiple nitrogen types, adjust ratios, or examine the effects of competition. given the few interesting results that are available, additional studies that track uptake kinetics and gene expression in mixed nitrogen nutrition experiments, and when plants compete would provide key information about the plasticity plants possess in uptake kinetics, and greatly advance the general understanding of plant nitrogen nutrition in all fields of plant sciences.  it is now recognized that plants regularly capture amino acids along with mineral sources of nitrogen, and it is generally assumed that amino acid nutrition enhances access to nitrogen and is a net fitness benefit to plants. the results presented above suggest that at concentrations that are similar to those found in natural soils, amino acids contribute relatively little to potential vegetative growth or reproductive output in a. thaliana relative to inorganic forms of nitrogen such as nitrate. in all experiments plants achieved lower performance when supplied with amino acids, and preferentially captured nitrate over amino acids when given nitrogen in mixture. furthermore, there was a negative interaction between amino acids and nitrate when plants were fed with mixed nitrogen nutrition. these plants had lower overall growth and potential reproductive output then plants fed a similar amount of a single type of nitrogen. this suggests some cost associated with and expanded nitrogen diet, and raises questions about why plants bother to capture amino acids from soil. faced with these curious results, we hypothesize that the importance of amino acid nutrition may be related to nitrogen competition, and this role may only be obvious in experiments that compare plants grown alone to those grown with neighbours. regardless of the outcome, examining this untested hypothesis will provide valuable data about whether and how plants adjust their nutrient uptake kinetics. 
access to multiple nitrogen types in plant communities has been argued to enhance plant performance, access to nitrogen and alter ecological interactions in ways that may promote species coexistence. while it is known that plants uptake amino acids from soil, long term studies that link amino acid uptake to measures of plant performance and potential reproductive effort are not typically performed. plant performance on mixed nitrogen was less than half the performance on equal amounts of any single nitrogen type. ecologists recognize that plants capture nitrogen in many chemical forms that include amino acids. when plants were supplied with two types of nitrogen in mixture at ratios between: -:  mm, again plants performed best when nitrate was present, and poorly when amino acids were mixed. we asked: why did a. thaliana capture amino acids when doing so resulted in a net cost? we argue that available data cannot yet answer this question, but hypothesize that access to lower quality forms of nitrogen may become important when plants compete. here, a series of experiments that link uptake of nitrate, glutamine or asparagine with lifetime reproductive effort in arabidopsis thaliana are reported. when plants were supplied with a single nitrogen type at concentrations from -  mm, the ranking of nitrogen types was nitrate > glutamine > asparagine in terms of the relative performance of plants. additionally, stable isotopes revealed that plants preferentially captured nitrogen types matching the hierarchy of nitrate > glutamine > asparagine. traits related to reproductive output were measured, as was the preference for each type of nitrogen. amino acid uptakearabidopsis thalianareproductive outputnitrogen partitioningnitrogen preferencesplant foraging comparing between the two experiments revealed that mixed nitrogen nutrition was a net cost to the plants.
 ecologists recognize that plants capture nitrogen in many chemical forms that include amino acids. access to multiple nitrogen types in plant communities has been argued to enhance plant performance, access to nitrogen and alter ecological interactions in ways that may promote species coexistence. however, data supporting these arguments have been limited. while it is known that plants uptake amino acids from soil, long term studies that link amino acid uptake to measures of plant performance and potential reproductive effort are not typically performed. here, a series of experiments that link uptake of nitrate, glutamine or asparagine with lifetime reproductive effort in arabidopsis thaliana are reported. nitrogen was offered either singly or in mixture and at a variety of combinations. traits related to reproductive output were measured, as was the preference for each type of nitrogen.  when plants were supplied with a single nitrogen type at concentrations from -  mm, the ranking of nitrogen types was nitrate > glutamine > asparagine in terms of the relative performance of plants. when plants were supplied with two types of nitrogen in mixture at ratios between: -:  mm, again plants performed best when nitrate was present, and poorly when amino acids were mixed. additionally, stable isotopes revealed that plants preferentially captured nitrogen types matching the hierarchy of nitrate > glutamine > asparagine. comparing between the two experiments revealed that mixed nitrogen nutrition was a net cost to the plants.  plant performance on mixed nitrogen was less than half the performance on equal amounts of any single nitrogen type. we asked: why did a. thaliana capture amino acids when doing so resulted in a net cost? we argue that available data cannot yet answer this question, but hypothesize that access to lower quality forms of nitrogen may become important when plants compete. amino acid uptakearabidopsis thalianareproductive outputnitrogen partitioningnitrogen preferencesplant foraging 