Plant, Cell and Environment (2011) 34, 525–534
doi: 10.1111/j.1365-3040.2010.02260.x
Constrained preferences in nitrogen uptake across plant
species and environments
pce_2260
525..534
LIXIN WANG1 & STEPHEN A. MACKO
Department of Environmental Sciences, University of Virginia, Charlottesville, VA 22904, USA
ABSTRACT
Knowledge of determining factors for nitrogen uptake preferences and how they are modified in changing environments are critical to understand ecosystem nitrogen cycling
and to predict plant responses to future environmental
changes. Two 15N tracer experiments utilizing a unique
differential labelled nitrogen source were employed in
both African savannas and greenhouse settings. The results
demonstrated that nitrogen uptake preferences were constrained by the climatic conditions. As mainly indicated by
root d 15N signatures at 1:1 ammonium/nitrate ratio, in the
drier environments, plants preferred nitrate and in the
wetter environments they preferred ammonium. Nitrogen
uptake preferences were different across different ecosystems (e.g. from drier to wetter environments) even for the
same species. More significantly, our experiments showed
that the plant progeny continued to exhibit the same nitrogen preference as the parent plants in the field, even when
removed from their native environment and the nitrogen
source was changed dramatically. The climatic constraint
of nitrogen uptake preference is likely influenced by
ammonium/nitrate ratios in the native habitats of the
plants. The constancy in nitrogen preference has important
implications in predicting the success of plant communities
in their response to climate change, to seed bank use and to
reforestation efforts.
Key-words: adaptation; C4 plants; isotope enrichments;
grass; Kalahari; nitrogen cycling; nitrogen preferences;
savannas; stable isotopes.
INTRODUCTION
Nitrogen (N) is the principal limiting nutrient for most terrestrial and aquatic ecosystems (Aber et al. 1989; Davidson
et al. 2007; Hobbie & Hobbie 2006; Howarth & Marino
2006) with ammonium (NH4+) and nitrate (NO3-) being the
Correspondence: L. Wang. Fax: +609 258 1436; e-mail: w.lixin@
gmail.com
1
Present Address: Department of Civil and Environmental Engineering, Princeton University, Princeton, NJ 08544, USA. E-mail:
[email protected]
© 2011 Blackwell Publishing Ltd
two major inorganic N forms available for plants (e.g.
Mahmood & Kaiser 2003). Recent studies reinforce the
idea that plants clearly prefer inorganic N forms over
organic forms in most systems (Harrison, Bol & Bardgett
2007; Ashton et al. 2008), and the optimization of the plant
preference of different forms of inorganic N presumably
enhances the survivorship and the fitness of plants. Even
though differences in the rates of NH4+ and NO3- uptake
have been documented by a number of physiological
studies (Olsson & Falkengren-Grerup 1995; Kronzucker,
Siddiqi & Glass 1997; von Wirén, Gazzarrini & Frommer
1997; Wallander et al. 1997; Garnett & Smethurst 1999) and
one ecosystem-scale study (Kahmen, Wanek & Buchmann
2008), the ecological origins and significance of N uptake
preference remain poorly understood. In a recent field
study conducted in Hawaiian tropical forests through a
novel 15N natural abundance tracer approach, it was found
that the plants switched N source from NO3- to NH4+ along
a gradient from drier to wetter sites (Houlton et al. 2007).
However, the biological basis of these changes and how
common the observed phenomena across different biomes
are unclear, for example, whether such a trend also occurs
in a much drier plant-soil gradient. Even less is known
about how such preferences will be altered in a changing
environment.
The assimilation of NH4+ is more energetically efficient
when compared with NO3-, because NH4+ can be directly
incorporated into glutamate via an NH4+ assimilation
pathway. Nitrate, on the other hand, must first be modified
via a reduction pathway before assimilation (Engels & Marschner 1995). However, NO3- is usually more available for
uptake in many ecosystems, owing to its higher mobility
(Brady & Weil 1999). Nitrate can be incorporated into
organic compounds in both root and leaf tissues whereas
NH4+ is only synthesized into amino acids in the root tissues
near the site of uptake to avoid toxic accumulation (Engels
& Marschner 1995). Either NH4+ or NO3- can dominate
the inorganic N pool of an ecosystem. For example, in most
mature undisturbed forests, the soil inorganic N pools
are dominated by NH4+ (Kronzucker et al. 1997). In wellaerated agricultural soils or other frequently disturbed sites,
NO3- is the principal inorganic N source (Brady & Weil
1999). In arid and semiarid ecosystems such as African
savannas, nutrient availability varies spatially and temporally, and nutrients are considered to be a major limiting
525
526 L. Wang and S. A. Macko
Figure 1. Geographic location (black dot) and vegetation structure of each study site along the Kalahari Transect. The grey area in the
map is the Kalahari sand sheet.
factor for plant growth when water limitation alleviates
(Scholes et al. 2002; Aranibar et al. 2004). The Kalahari
Transect (KT) in southern Africa traverses a dramatic
aridity gradient through Zambia, Botswana, Namibia, and
the Republic of South Africa and is essentially composed
of homogeneous soils – the deep Kalahari sands (Fig. 1)
(Shugart et al. 2004; Wang et al. 2007). The rainfall variability along the KT ranges from less than 200 mm mean annual
precipitation (MAP) in southwest Botswana to over
1000 mm MAP in the north (i.e. western Zambia) (Shugart
et al. 2004). Therefore, the KT provides ideal conditions
(homogenous soils and gradients in rainfall) for studying, at
subcontinent scales, the association between N uptake preferences and aridity, without confounding soil effects. This
uniquely designed N uptake study was accomplished by
using 15N as a differential labelled source of N, in both
greenhouse and field-based setting, to test two hypotheses.
The first is that N preferences are constrained by climatic
conditions of the native habitat of the plants. Following the
observations of Houlton et al. (2007) in Hawaiian rainforests, we hypothesized that plants prefer NO3- in the drier
environments, and plants prefer NH4+ in the wetter environments.We further hypothesized that the climatic constraints
on N uptake preference is influenced by the NH4+/NO3ratios in the habitats; and that – the second hypothesis –
plants may inscribe the adaptation of the N uptake preference in their progeny. All greenhouse and field plants were
fertilized with NH4NO3. There were two treatments, one
with 14NH415NO3 and the other with 15NH414NO3, allowing
for direct monitoring of N uptake preferences. Whenever
possible, a control (without fertilizer addition) was also
utilized.
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 525–534
Plant nitrogen preference 527
Table 1. Field characteristics of five study sites along the Kalahari Transect and species list for the field and greenhouse experiments
Location
Elevation
(m)
Mean annual
rainfall,
mm/year
Tshane
24.17°S, 21.89°E
1115
Ghanzi
21.65°S, 21.81°E
Maun
Pandamatenga
Mongu
Greenhouse experiment
Field experiment
365
Enneapogon cenchroides,
Eragrostis lehmanniana
Schmidtia pappophoroides,
Eragrostis lehmanniana
1125
424
Schmidtia pappophoroides,
Eragrostis lehmanniana
Stipagrostis suniplumis, Eragrostis
lehmanniana
19.92°S, 23.59°E
929
460
18.66°S, 25.50°E
1082
698
Schmidtia pappophoroides
Leptocarydion vulpiastrum
Pogonarihria squarrosa
15.44°S, 23.25°E
1076
879
Eragrostis spp.
MATERIALS AND METHODS
Greenhouse experiment
For the greenhouse experiment, a total of six different
grass species from four sites of their native habitat along
the KT precipitation gradient (Fig. 1) were used in this
study (Table 1). Seeds of four grass species were collected in August 2004, and seeds of two other grass species
(Schmidtia pappophoroides Steud. ex J.A. Schmidt from
Pandamatenga and Eragrostis spp. from Mongu) were collected in March 2005. In Mongu (897 mm MAP), the
wettest area in this mega transect, seeds of Eragrostis spp.
was collected. In Pandamatenga (698 mm MAP), the
second wettest area in the transect, seeds of S. pappophoroides, Pogonarihria squarrosa (Licht, ex Roem. & Schult.)
Pilg. and Leptocarydion vulpiastrum Stapf were collected.
In Ghanzi (424 mm MAP), Botswana, a relatively dry site
with intermediate rainfall conditions, seeds of S. pappophoroides and Eragrostis lehmanniana Nees were collected. In
Tshane (365 mm MAP) of southern Botswana, the driest
site of this transect (Table 1), Enneapogon cenchroides
(Licht.) Roem. & Schult. ex C.E. Hubbard and E. lehmanniana were collected. Based on our own field observations,
the number of grass species increased from the very dry
area to wetter area and decreased from wetter area to the
wettest area (Table 2, Wang et al. 2010). The number of
species used in the greenhouse and field experiments generally followed the pattern of species distribution in the
field. The greenhouse experiment was conducted at the
University of Virginia from February to October 2005.
The two additional grass species (S. pappophoroides from
Pandamatenga and Eragrostis spp. from Mongu) were
grown in the greenhouse from February to June 2006.
The seeds were initially germinated in pans.The seedlings
were then transferred into plastic pots (one individual per
pot) containing commercial sand. Plants were equally wellwatered during their germination and growth (volumetric
soil water content was between 20 and 40%). During
an initial two-week period of adjustment after transferring
from germination pans to individual pots, a low concentration (300 ppm) commercial soluble fertilizer [Peters
N (2.1% NO3-, 17.9 urea)-P (phosphate)-K (potash):
Schmidtia pappophoroides
Schmidtia pappophoroides,
Leptocarydion vulpiastrum,
Panicum maximum, Digitaria spp.
20–20-20] was applied to facilitate survival. A couple of
weeks after adjustment from transplanting, a 15N-labelled
fertilizer (NH4NO3, 50‰ in 15N) was applied to each individual plant to establish an N concentration of 15 mg N/g
dry soil. The total amount of N applied as fertilizer was
comparable with the abundance of mineral N at the African
field locations (Feral et al. 2003; Wang et al. 2007). The 15Nlabelled fertilizer solution was evenly sprayed around the
base of each plant (2 cm radius) at a very slow rate to
prevent diffusion of the fertilizer exceeding the 2 cm radius,
and the solution infiltrated about 20 cm of the potting sand
(the roots were about 20–30 cm deep and the root distributions were similar for all the species). The 15N-labelled
fertilizer solution was applied twice in 48 h. The two
treatments (NH415NO3 and 15NH4NO3) received the same
amount of total N fertilizer. In order to distinguish the
uptake preferences, the 15N was labelled at different
Table 2. The grass richness data of the four sites along the
Kalahari Transect
Location
Species
richness
Tshane
2
Eragrostis lehmanniana
Schmidtia pappophoroides
Ghanzi
3
Urochloa brachyura
Stipagrostis suniplumis
Schmidtia pappophoroides
Pandamatenga
8
Panicum maximum
Aristida stipitata
Megaloprotachne albescens
Pogonarihria squarrosa
Tricholaena monachne
Digitaria species
Heteropogon contortus
Urochloa brachyura
Mongu
2
Eragrostis spp.
Unidentified grass
Species name
The data was modified from a field survey conducted in the wet
season 2005. The species richness is based on the number of species
from 20 1 ¥ 1 m2 plots from each site and the details of field
sampling can be found in Wang et al. (2010).
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 525–534
528 L. Wang and S. A. Macko
locations in the N compounds (15NH4+ versus 15NO3-). NH4+
and NO3- have the same molar concentrations because of
the chemical composition of NH4NO3. If plants have any
preference for either NH4+ or NO3-, we could measure it
through the differences in d 15 N signatures between NH4+
labelled treatment and NO3- labelled treatment. The comparisons were always between roots or between leaves
(e.g. no cross-comparisons of roots and leaves). If the d 15N
is higher in the NO3- labelled treatment than the NH4+
labelled treatment, the plants prefer NO3-; if the d 15N is
higher in the NH4+ labelled treatment, the plants prefer
NH4+.
Small isotopic fractionation of NH4+ and NO3- uptake
with high external N concentrations has been observed in
plants grown in nutrient solutions (Högberg et al. 1999;Yoneyama et al. 2001). Because equal molar NH4+ and NO3- was
applied to all the species and to all the sites, the fractionation was assumed to have minimal effects on the observed
d15N signature patterns. Many studies (e.g. Drew & Saker
1978; Jackson, Manwaring & Caldwell 1990; Wang, Mou &
Jones 2006) have shown that plants can respond to nutrient
addition within hours of applications. Therefore short application duration (one injection per 24 h interval and 48 h
in total) was employed in this study, which can minimize
biases caused by the potential N transformation processes
in the potting sand. Four individuals were used for each
species ¥ treatment combination (three individuals of E.
lehmanniana from Tshane were used for 15NH4NO3 treatment, owing to lower seedling availability). Three individual
plants of each species were used as controls, and were
grown without fertilizer (except the low initial soluble fertilizer to facilitate survival) or labelled 15N additions. To
evaluate the effect of higher soil N content on N preference,
double amount of fertilizer (but the same 15N signature)
was applied to P. squarrosa and L. vulpiastrum in addition
to the field-level N treatment mentioned above. All grasses
were harvested 24 h after the second fertilizer application.
The roots were carefully rinsed with tap water to remove
excess N fertilizer that was not taken up. Leaves and
roots from each individual were separated and stored in
paper bags.
Field experiment
In January and February 2006, field 15N labelling and uptake
experiment was carried out in four locations along the KT
where the grass seeds were obtained. The main objective of
the field experiment was to test the plant N uptake preference in situ and to explore the mechanisms of N uptake
preference by comparing field (parents) and greenhouse
(F1 generation) results. The species selected were essentially the same as those used in the greenhouse experiment
(Table 1) and the locations were similar though not exactly
the same because of field logistics. The non-exact match of
field and greenhouse sites may not be ideal, however, three
sites overlapped between the field and greenhouse experiments and these overlaps included both dry and wet locations – the major contrast in this study. The field N uptake
experiment was conducted in exactly the same way as the
greenhouse experiment except a higher 15N signature for
both NH4+ and NO3- (100‰ 15NH4NO3 and NH415NO3) was
used. The 15N-labelled fertilizer solution was evenly sprayed
around the base of each plant (2 cm radius) at a very slow
pace and the water infiltrated about 10–20 cm of the soil.
The root distributions were similar for all the species but
the root sizes varied depending on species. We chose
individuals of similar sizes for 15NH4 and 15NO3 treatments
to avoid complications because of root size differences.
The 15N-labelled fertilizer solution was applied twice in
48 h before harvesting. At Pandamatenga, control plants
(without nutrient or water addition) of S. pappophoroides,
Panicum maximum and L. vulpiastrum were collected and
analysed for comparison.
Chemical analysis
All samples were then oven-dried at 60 °C for 72 h. After
drying, foliar and root samples were ground to powder.
Nitrogen isotope analysis was performed using a GV
Micromass Optima isotope ratio mass spectrometer
(IRMS) coupled to a Carlo Erba elemental analyser (EA).
The 15N content of the plants was reported in the conventional notation:
δ 15 N (‰) = ⎡⎣( 15 N
14
N )sample
( 15 N 14 N )standard − 1⎤⎦ × 1000
where (15N/14N)sample is the N isotope ratio of samples, and
(15N/14N)standard is the N isotope ratio of the standard material. The standard for N stable isotopes is atmospheric
molecular N. Reproducibility of the measurements was
approximately 0.2‰.
Statistical analysis
The existence of N uptake preference was determined by
the d 15N difference between 15NH4+ and 15NO3- treatment
in either the roots or the leaves. Considering the different
N assimilation locations for NO3- and NH4+ (both roots
and leaves for NO3- versus roots only for NH4+) and the
short duration of the experiments, only root d15N differences between 15NO3- and 15NH4+ treatment were used to
indicate NH4+ preference whereas both the foliar and root
d15N differences were used to indicate NO3- preference. A
one-way ANOVA and Tukey post hoc test (at a = 0.05 significance level) was used to evaluate the significance of the
differences detected in the roots or leaves for all species
from each site. To better visualize the results and compare
parent versus progeny N uptake preference, the degrees
and forms of N uptake preference at each site were summarized against MAP of each site for both field and greenhouse settings (e.g. Figs 2e and 4e). The degrees and forms
of N uptake preference were indicated by 15N signature
differences between 15NH4+ and 15NO3- treatment either in
root (for NH4+ and NO3- preference) or in leaves (for NO3preference). For NO3- preference, if the 15N signatures
in both the root and foliar samples were significantly
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 525–534
Plant nitrogen preference 529
different between 15NH4+ and 15NO3- treatment, the larger
difference value was used to indicate the maximum NO3preference. In the summary figures (e.g. Figs 2e and 4e),
if the difference (15NO3- - 15NH4+) was a positive value,
the plant preferred NO3-; if negative, the plant preferred
NH4+. A value of 0 indicated that no significant preference
was detected.
RESULTS
For the greenhouse experiment, the individuals in relatively
dry areas (E. cenchroides and E. lehmanniana from Tshane,
and S. pappophoroides and E. lehmanniana from Ghanzi)
exhibited a preference for NO3- (Fig. 2a,b,e), indicated by
higher foliar d 15N signatures in 15NO3- treatment than in
15
NH4+ treatment. Whereas others in the wetter habitats (S.
pappophoroides, P. squarrosa and L. vulpiastrum from Pandamatenga and Eragrostis spp. from Mongu) had a preference for NH4+, indicated by higher root d 15N signatures in
15
NH4+ treatment than in 15NO3- treatment (Fig. 2c,d,e). For
the two species (P. squarrosa and L. vulpiastrum) with additional treatment of higher concentrations of NH4NO3 (but
the same 15N signature), their uptake preferences did not
change (Fig. 3).
The field experiment generally showed the same trends
with one exception. The individuals in relatively dry areas
(S. pappophoroides and E. lehmanniana from Tshane, and
S. suniplumis from Ghanzi) exhibited a preference for
NO3- (Fig. 4a,b,e), indicated by higher d 15N signatures in
15
NO3- treatment than in 15NH4+ treatment either in roots
(S. pappophoroides in Tshane and S. suniplumis in Ghanzi)
or in leaves (E. lehmanniana in Tshane). The exception
was E. lehmanniana from Ghanzi, which did not show any
preference (Fig. 4b). This was different from what was
observed in the greenhouse experiment for this species of
this site (Figs 2b and 4b). The individuals in the wetter
areas (S. pappophoroides, P. maximum, L. vulpiastrum and
Digitaria spp. from Pandamatenga) had a preference for
NH4+, indicated by higher root d 15N signatures in 15NH4+
treatment than in 15NO3- treatment (Fig. 4d,e). The individuals (S. pappophoroides) in Maun (with an intermediate
rainfall level) did not show preference for either NH4+ or
NO3- (Fig. 4c,e).
DISCUSSION
The hypothesis that N preferences are influenced by climatic conditions of the native habitat of the plants is supported by the data from both the greenhouse and field N
uptake experiments. In both settings, plants showed preference for NO3- in the drier environments and showed
preferences for NH4+ in the wetter environments. More convincingly, the species (S. pappophoroides) that appeared
in different environments (e.g. from drier to wetter environments) showed different N uptake preferences ranging from NO3- in drier environments to NH4+ in wetter
environments in the greenhouse condition (Fig. 2e); and
ranging from NO3- in the driest site to no preference in the
intermediate site and then to NH4+ in the wettest site in the
field condition (Fig. 4e). The variation in N preferences
clearly tracked the environmental condition under which
they originally grew.
The absorbed N will eventually end up in the leaf/shoot
issue and the foliar 15N signature is a good indicator for N
uptake preference. However, our method of using both
leaf and root tissue 15N signatures to indicate N uptake
preference is valid for this particular experimental design
based on the following reasons. NH4+ is only synthesized
into amino acids in the root tissues (Engels & Marschner
1995). It is also known that up to 95% (with a mean of 58%)
of the labelled 15N remained in root systems of various
species after 48 h of 15N labelling in an earlier 15N labelling
experiment with similar experimental setup (Wang, Mou &
Jones 2006). In this study, we limited the N labelling duration to 48 h to minimize the effect of N mineralization and
nitrification on 15N signatures. Therefore the relatively short
labelling duration may not be long enough to allow assimilated N to transport from root tissues to shoot tissues and it
is not surprising to see that the NH4+ preference and some
of the NO3- preference (S. pappophoroides from Tshane,
and S. suniplumis from Ghanzi) were shown only in the root
15
N signatures.
The climatic switch of plant N uptake preference is likely
determined by the NH4+/NO3- ratios of the habitats. Several
lines of evidences support this argument. Firstly, previous
field observations along the KT showed that NH4+/NO3ratios are higher at the wettest end (Supporting Information
Fig. S1) (Aranibar 2003; Feral et al. 2003). This NH4+/NO3ratio gradient is likely controlled by both biological and
hydrological factors. In the wetter end, there are higher
decomposition rates (more NH4+ availability) and more
nitrate leaching (Scanlon & Albertson 2003), leading to a
higher NH4+/NO3- ratio. Our observed N uptake preference
switches between the dry and wet habitats match the NH4+/
NO3- ratio gradient of the KT. Secondly, there are several
laboratory and field observations from distinct geographic
regions showing NH4+/NO3- ratios of the sites affect plant
N uptake preferences. For example, by using kinetic and
compartment-analysis techniques with the radiotracer 13N to
compare the efficiency of N acquisition from NH4+ and NO3sources in the seedlings of Picea glauca (white spruce) of
western Canada, it was found that the uptake of NH4+ is 20
times higher than that of NO3- from an equimolar solution;
cytoplasmic concentrations of NH4+ are 10 times higher than
that of NO3- (Kronzucker et al. 1997). Similarly, it was found
that the N uptake by Pinus sylvestris (Scotch pine) in Scandinavia from a 15N-labelled solution of NH4+ is much higher
(about 10-fold) than from a 15N-labelled solution of NO3(Wallander et al. 1997). The chief form of inorganic N available for P. glauca and P. sylvestris, in their native habitats, is
NH4+. On the other hand, Populus tremuloides (trembling
aspen) and Pinus contorta (lodgepole pine), two early
successional species, were found to show a very high NO3utilization rates at high external NO3- concentrations, and
their common habitats are disturbed sites where available
N is predominant by NO3- (Min et al. 1998). In addition,
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 525–534
530 L. Wang and S. A. Macko
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 525–534
Plant nitrogen preference 531
Figure 2. Greenhouse results for the d 15N signature in plant roots and leaves for species from Tshane (a), Ghanzi (b), Pandamatenga
(c) and Mongu (d) along the Kalahari Transect (KT). Dashed lines separate species at one site. The different capital letters indicate
significantly different means between treatments (NH4+, NO3- and control) for either root or foliar samples for each species at each site
using one-way analysis of variance and Tukey post hoc test (P < 0.05). (e) Summary of changes in plant nitrogen uptake preference along
the KT in greenhouse study. The x-axis identifies the different locations by mean annual precipitation (MAP). The y-axis is the d 15N
signature difference between 15NO3- and 15NH4+treatments for the species at each location. The error bars are standard deviations. If the
difference is a positive value, the plant prefers NO3-; if negative, the plant prefers NH4+. A value of 0 indicates that no preference is
detected. The larger difference is used to indicate the maximum preference if the 15N signature in both the root and foliar samples are
significantly different between 15NH4+ and 15NO3- treatments for NO3- preference, and for detailed explanations please refer to Materials
and Methods section in the text.
Houlton et al. (2007) found that from drier sites to wetter
sites the plants switch N source from NO3- to NH4+ and such
switch is in accordance with the NH4+/NO3- ratio changes
in Hawaii tropical forests. Based on our direct monitoring
of N uptake preference and evidences from previous work,
we think it is probably a ubiquitous phenomenon that soil
NH4+/NO3- ratio in plants’ native habitat determines plant N
uptake preference across different ecosystems.
More significantly, through the comparison of the
summary of field results (Fig. 2e) and of the greenhouse F1
generation results (Fig. 4e), our observations suggest that
the plants may maintain the adaptation in N preference, at
least for the first generation. The field experiments demonstrated that plants along the KT showed N uptake preferences that tracked the environmental conditions. When
seeds from each location (with different environmental
conditions) were collected, germinated and grown in the
Figure 3. Greenhouse results for the d 15N signatures in plant
roots and leaves for species from Pandamatenga. A doubled
mineral N concentration with the same d 15N enrichment is used
for these two species. The different capital letters indicate significant differences in the means among treatments (NH4+, NO3- and
control) for either root or foliar samples for each species using
one-way analysis of variance and Tukey post hoc test (P < 0.05).
greenhouse with equal amounts of water and N being
applied (which were different from their field conditions),
the plants showed the same N preferences as the parent
plants did in the field (Figs 2e and 4e), indicating that plants
appear to maintain the parental adaptation (at least for the
F1 generation) with respect to N uptake preference. This
constancy in N preference is further supported by the additional greenhouse observations that when higher concentrations of NH4NO3 were applied to P. squarrosa and L.
vulpiastrum, the uptake preferences for the form of N did
not change (Fig. 3). Although the current findings do not
distinguish genetic effects from maternal effects for the
‘imprint’ of N uptake preference, such consistent results
imply that, for example, during climate change scenarios,
plant communities (at least for herbaceous plants) may
keep their original physiological traits and pass them on
to future generations. Longer-term studies of plant communities are clearly necessary to infer the physiological
responses to climate change. The similar responses in the
greenhouse and the field settings also indicate that the
observed patterns are not caused by bonding differences of
soil particles and NH4+ among sites as the soil substrate was
the same in the greenhouse experiment.
The ammonium nitrate fertilizer with differential labelling treatment provides a unique and direct way to test N
uptake preference as shown in this study, though the preference observations were only tested for a 1:1 ratio. Previous studies showed that NO3- uptake can be depressed by
the supply of NH4+ (e.g. Lee & Drew 1989), and the negative
effect of NH4+ supply on NO3- uptake depends on the utilization of energy during NH4+ assimilation or the inhibitory
effect of assimilatory products rather than on NH4+ per se
(Engels & Marschner 1995). However, the NH4+ suppression effect on NO3- uptake was reported to be minimum
when NH4+ concentration is under 1 mM (Breteler &
Siegerist 1984; Engels & Marschner 1995). In the current
experiment, the labelling solution was only around
0.02 mM, therefore the NH4+ suppression effect on NO3uptake should be small, if any.
Amino acids and dissolved organic nitrogen (DON) are
other potential nitrogen sources for plant uptake and
plant uptake of amino acids have been found in various
boreal ecosystems (e.g. Schimel & Chapin 1996; Persson &
Nasholm 2001). In arid and semi-arid environments,
however, there are very limited data on plant DON and
amino acid uptake or even on DON and amino acid
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 525–534
532 L. Wang and S. A. Macko
Figure 4. Field results for the d 15N signature in plant roots and leaves for species from Tshane (a), Ghanzi (b), Maun (c) and
Pandamatenga (d) along the Kalahari Transect (KT). Dashed lines separate species at one site. The different capital letters indicate
significantly different means between treatments (NH4+, NO3- and control) for either root or foliar samples for each species at each site
using one-way analysis of variance and Tukey post hoc test (P < 0.05). (e) Summary of changes in plant nitrogen uptake preference along
the KT in field study with the same layout as Fig. 2e.
distributions. The water diffusivity in dry soils can range
between 10-6 and 10-7 m2/year (for values of soil moisture
typical of the root zone), such low diffusivity will probably
limit the movement of DON and amino acid and therefore the plant uptake of DON and amino acid in arid and
semi-arid environments. Although this study did not assess
the role that either DON or amino acid plays in these
ecosystems, it is worthwhile to explore their potential roles
in future research.
In summary, this study has illustrated a climatic constraint on the preferential uptake of the different forms of
inorganic N by plants and indicated that the N uptake
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 525–534
Plant nitrogen preference 533
preference is likely influenced by the NH4+/NO3- ratios
in the native habitats of the plants. From an evolutionary
point of view, it supports the local adaptation concept
(Endler 1986; Chapin 1988). It is also along the line that
the most abundant species rely on the most abundant N
forms as shown in the study of McKane, Grigal & Russelle
(1990). In addition, the results suggest that ‘imprinting’ of
such preference exists in seeds. It showed that plant seeds
retain the adaptation towards the N uptake preference of
their parents, even when the abundances of NO3- and
NH4+ changed. Practically, the maintenance of plant N
uptake preference across generations provides an underlying mechanism explaining why, for example, a majority
of the replanted conifer species in western Canada failed
to survive as observed in a study of Kronzucker et al.
(1997), that is, the newly replanted trees have the potential
genetic imprint of NH4+ uptake preference whereas the
soil in the new environment after clear cutting is dominated by NO3-. The maintenance of plant N uptake preference also has implications to seed bank use.
ACKNOWLEDGMENTS
The project was funded by NASA-IDS2 (NNG-04GM71G). We greatly appreciate the assistance in seedlings
identification from Chris Feral and Kebonyethata Dintwe.
Ms. Wendy Crannage provided tremendous help in the
greenhouse maintenance. We also appreciate the teamwork
and field assistance from Lydia Ries, Thoralf Meyer and
Paolo D’Odorico at University of Virginia, Billy Mogojw at
Harry Oppenheimer Okavango Research Center, University of Botswana. We thank Dr Howard Epstein for the
thoughtful discussion at the beginning of the experimental
design. The clarity of this manuscript is significantly
improved by comments from Dr Manuel Lerdau, Dr Jin
Wang, the ecology and isotope geochemistry discussion
groups in Department of Environmental Sciences of the
University of Virginia and Dr Lars Hedin from Princeton
University. We are grateful for the comments from two
anonymous reviewers and editor Dr Werner Kaiser.
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Received 29 July 2010; received in revised form 28 September 2010;
accepted for publication 6 October 2010
SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Figure S1. NH4+ and NO3- abundance (mmol g-1) (A) and
NH4+/NO3- ratios (B) of the study sites along the Kalahari
Transect based on field nutrient data of Feral et al. (2003).
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the
article.
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 525–534