Skip to main content

Nitrogen uptake strategies of mature conifers in Northeastern China, illustrated by the 15N natural abundance method

Abstract

Background

Conifers partition different N forms from soil, including ammonium, nitrate, and dissolved organic N (DON), to sustain plant growth. Previous studies focused on inorganic N sources and specific amino acid forms using 15N labelling, but knowledge of the contribution of DON to mature conifers’ N uptake is still scarce. Here, we quantified the contribution of different N forms (DON vs. NH4+ vs. NO3) to total N uptake, based on 15N natural abundance of plant and soil available N, in four mature conifers (Pinus koraiensis, Pinus sylvestris, Picea koraiensis, and Larix olgensis).

Results

DON contributed 31%, 29%, 28%, and 24% to total N uptake by Larix olgensis, Picea koraiensis, Pinus koraiensis, and Pinus sylvestris, respectively, whereas nitrate contributed 42 to 52% and ammonium contributed 19 to 29% of total N uptake for these four coniferous species.

Conclusions

Our results suggested that all four conifers could take up a relatively large proportion of nitrate, while DON was also an important N source for the four conifers. Given that DON was the dominant N form in study soil, such uptake pattern of conifers could be an adaptive strategy for plants to compete for the limited available N sources from soil so as to promote conifer growth and maintain species coexistence.

Background

Conifers are the main afforestation species in Northeast China and play indispensable roles in ecological services such as timber production and regulating regional climate (Zhou et al. 2020). Conifers use different N forms from soil as their primary N sources, including ammonium, nitrate, and dissolved organic N (DON) (Näsholm et al. 2009; Orwin et al. 2011). Given the relatively lower soil available N concentrations in temperate climates (Zhu et al. 2019), conifers would partition limited available N sources to sustain plant growth (Kronzucker et al. 1997; Gao et al. 2020), which is an important mechanism underlying species coexistence (McKane et al. 2002). However, it remains unclear how conifers partition limited N resource due to the complex interactions between plant N uptake and a variety of environmental conditions (Britto and Kronzucker 2002; Houlton et al. 2007).

Previous studies mainly investigated the inorganic N sources (ammonium and nitrate) for conifer N uptake using 15N labelling (Zhu et al. 2019; Gao et al. 2020), in which conifers took up more nitrate in in situ experiments and took up more ammonium in hydroponic experiments (Kronzucker et al. 1997; Boczulak et al. 2014; Li et al. 2015; Liu et al. 2017). However, it remains unclear to what extent conifers can utilize organic N. Previous experiments have studied the contribution of specific amino acid forms using 15N- and 13C-labelled glycine, arginine, or glutamine (Öhlund and Näsholm 2004; Persson et al. 2006; Zhu et al. 2019). Results from such work may not represent the uptake patterns of DON by mature conifers because these amino acids generally only account for a small fraction of organic N pools (Yu et al. 2002; Andersson and Berggren 2005). Although DON is widely accepted as a potentially important N source (Näsholm et al. 2009; Andersen et al. 2017), the knowledge of the contribution of DON to N uptake by mature conifers is still limited.

Natural abundance of 15N (δ15N) in plants and soil can provide integrated information related to plant-soil physiological processes (Phillips and Gregg 2003; Houlton et al. 2007; Craine et al. 2009). Recently, the development of methods for determining the δ15N of ammonium and nitrate provided the possibility to comprehensively study the δ15N of plants and soil extractable N (Liu et al. 2014; Zhang et al. 2015). Combined with isotopic mixing models, the 15N natural abundance method has been used to quantify the contribution of different N forms (DON, ammonium, and nitrate) to total N uptake based on the δ15N in foliage and soil extractable N in several previous studies (Houlton et al. 2007; Takebayashi et al. 2010; Zhang et al. 2018). The analysis of δ15N in foliage and different N forms in soil could assess the contribution of DON to coniferous species in a relatively convenient and practical approach (Peri et al. 2012; Zhang et al. 2018).

In this study, we used 15N natural abundance method to study the N uptake strategy of the four main conifers used for afforestation in Northeastern China, specifically Pinus koraiensis, Pinus sylvestris, Picea koraiensis, and Larix olgensis. We compared the results with our previous in situ labelling studies of these four conifers (Zhou et al. 2020). Our major objectives were (1) to explore the contribution of different N forms to mature conifers in the field and (2) to test whether the N uptake strategies are similar if assessed by 15N natural abundance or by in situ labelling methods.

Methods and Materials

Study site

This study was conducted at the Mengjiagang Forest Farm in Huanan County, Heilongjiang Province in Northeast China (46°25′N, 130°42′E, elevation 170–575 m). The mean annual temperature was 2.7 °C, and annual precipitation averaged 550 mm. There are hundreds of shrub and herb species and more than 20 tree species in the study area. In this study, we selected coniferous plantations of three evergreen species, Pinus koraiensis, Pinus sylvestris, and Picea koraiensis, and one deciduous species, Larix olgensis. These conifers are the dominant plantation types and indispensable for timber production. These four conifers are associated with ectomycorrhizal fungi (Guo et al. 2008). The selected trees were about 40 years old and almost 20 m high. Soils are mainly dark brown soil, with pH values ranging from 5.5 to 5.8 (Zhou et al. 2020). The total N concentrations of bulk soil and foliage ranged from 0.38 to 0.39% and from 1.4 to 2.1%, and total C concentrations of bulk soil and foliage ranged from 4.93 to 5.38% and from 46.4 to 49.3%, respectively (Zhou et al. 2020).

Sample collection

In each plantation, we selected five conifers with similar diameter at breast height. To avoid root intermingling, the distance between each tree was greater than 10 m, and there was no canopy overlap between each other. In July 2019, 20–50 fully developed leaves were collected for each tree. Mineral soil samples within 1.5 m from the trunk of each tree were collected with a 6 cm inner diameter auger and divided into two layers (0–10 and 10–20 cm). The Oa+e layer was removed before mineral soil was collected and restored after completion. Samples from eight soil cores in each plot were mixed to one composite sample by different soil depth, and then passed through a 2 mm sieve to remove plant materials and rocks.

Chemical analyses

Within 6 h after sampling, 10 g fresh soil was extracted with 2 M KCl solution in a soil/solution ratio of 1:4. The extraction was frozen at −36 °C until concentration and isotopic analysis. Leaf samples were dried at 65 °C to constant weight, and another subsample of soil was air-dried at room temperature to measure the δ15N and C, N concentrations by elemental analyzer-isotope ratio mass spectrometry (Elementar Analysen Systeme GmbH, Germany; IsoPrime100, IsoPrime Limited, UK).

The concentrations of ammonium and nitrate in the extracts were quantified by colorimetric determination using a continuous chemical analyzer (SmartChem200, Rome, Italy). Total dissolved N (TDN) was converted to nitrate by alkaline persulfate digestion (Doyle et al. 2004; Knapp et al. 2005), and then measured by colorimetric determination using a continuous chemical analyzer. Extractable DON was estimated as the difference between the concentrations of TDN and DIN (Takebayashi et al. 2010; Zhang et al. 2018). The δ15N of nitrate was determined by “the denitrifier method,” in which NO3 is converted to N2O by the cultured denitrifying bacteria that lack N2O reductase activity (Liu et al. 2014). The δ15N of TDN was measured as nitrate by alkaline persulfate digestion. The δ15N of ammonium was measured using the micro-diffusion method followed by alkaline hypobromite (BrO) oxidation and hydroxylamine (NH2OH) reduction (Zhang et al. 2015). Stable N isotopic signatures of the produced N2O were analyzed using a continuous flow isotope ratio mass spectrometer (IsoPrime 100, IsoPrime Limited, UK) connected to a cryofocusing unit (Trace Gas Preconcentrator, Isoprime Limited, UK). Several compounds were used as references: IAEA N3, USGS 32, USGS 34, and USGS 35 for nitrate and IAEA N1, USGS 25, and USGS 26 for ammonium. The analytical precision for δ15N was less than 0.3‰. The δ15N of DON was calculated using the following mass and isotopic balance equation:

$$ {\delta}^{15}{\mathrm{N}}_{\mathrm{DON}}=\left({\delta}^{15}{\mathrm{N}}_{\mathrm{TDN}}\times \left[\mathrm{TDN}\right]-{\delta}^{15}{\mathrm{N}}_{\mathrm{N}\mathrm{O}3-}\times \left[{{\mathrm{N}\mathrm{O}}_3}^{-}\right]-{\delta}^{15}{\mathrm{N}}_{\mathrm{N}\mathrm{H}4+}\times \left[{{\mathrm{N}\mathrm{H}}_4}^{+}\right]\right)/\left[\mathrm{DON}\right] $$

Isotopic mixing model

We used two types of isotopic mixing models, as described by Houlton et al. (2007), to estimate the proportional contribution (f) of different N forms to plant N uptake.

Considering the possible isotope (15N) fractionation during plant N uptake, previous studies had shown that it may cause approximately 2‰ of 15N discrimination between root and shoots (Dawson et al. 2002; Houlton et al. 2007). In addition, relative to nonmycorrhizal plants, the ectomycorrhizal fungi may impart additional fractionation of 8‰ or less (Hobbie and Colpaert 2003; Craine et al. 2009; Zhu et al. 2019). Therefore, we assumed a combined isotopic effect of 10‰ in this study. We assumed that plants are characterized by a steady state between plant N uptake and losses through root decay belowground and leaf fall aboveground (Houlton et al. 2007). If below- and above-ground N losses are equal, the δ15N of foliage will be 5‰ (half of the isotopic effect, 10‰) lower than the plant N sources (Houlton et al. 2007; Zhang et al. 2018). Moreover, to assess the uncertainty caused by isotope fractionation, we tested the sensitivity of the model if the isotopic effect varied from 4 to 12‰ (correction of 2–6‰). First, we assumed that plants could only utilize two N forms: ammonium and nitrate. The two end-member calculation takes on the following equations, and the *δ15Nfoliage is the measured foliage corrected for the internal isotope effect:

$$ 1={f}_{\mathrm{NO}3-}+{f}_{\mathrm{NH}4+} $$
$$ ^{\ast }{\delta}^{15}{\mathrm{N}}_{\mathrm{foliage}}={\delta}^{15}{\mathrm{N}}_{\mathrm{foliage}}+5{\mbox{\fontencoding{U}\fontfamily{wasy}\selectfont\char104}} $$
$$ ^{\ast }{\delta}^{15}{\mathrm{N}}_{\mathrm{foliage}}={f}_{\mathrm{N}\mathrm{O}3-}\times {\delta}^{15}{\mathrm{N}}_{\mathrm{N}\mathrm{O}3-}+{f}_{\mathrm{N}\mathrm{H}4+}\times {\delta}^{15}{\mathrm{N}}_{\mathrm{N}\mathrm{H}4+} $$

Second, another set of calculations included DON as a potential N source for plants in the following equations:

$$ 1={f}_{\mathrm{NO}3-}+{f}_{\mathrm{NH}4+}+{f}_{\mathrm{DON}} $$
$$ ^{\ast }{\delta}^{15}{\mathrm{N}}_{\mathrm{foliage}}={\delta}^{15}{\mathrm{N}}_{\mathrm{foliage}}+5{\mbox{\fontencoding{U}\fontfamily{wasy}\selectfont\char104}} $$
$$ ^{\ast }{\delta}^{15}{\mathrm{N}}_{\mathrm{foliage}}={f}_{\mathrm{N}\mathrm{O}3-}\times {\delta}^{15}{\mathrm{N}}_{\mathrm{N}\mathrm{O}3-}+{f}_{\mathrm{N}\mathrm{H}4+}\times {\delta}^{15}{\mathrm{N}}_{\mathrm{N}\mathrm{H}4+}+{f}_{\mathrm{DON}}\times {\delta}^{15}{\mathrm{N}}_{\mathrm{DON}} $$

In addition to the above three end-member calculations, we used an alternative approach to estimate the contribution of DON to plant N uptake. First, we assumed that the relative contributions of ammonium and nitrate to total N uptake of these four conifers (fNO3− and fNH4+) from the in situ labelling experiment were correct (Zhou et al. 2020). Second, we incorporated DON as a potential N source for plants and calculated the relative contribution of different N forms by three new end-member calculations according to the following equations:

$$ 1={f}_{\mathrm{NO}3-}+{f}_{\mathrm{NH}4+}+{f}_{\mathrm{DON}} $$
$$ {f}_{\mathrm{NO}3-}/{f}_{\mathrm{NH}4+}=1.4\ \mathrm{to}\ 3.3 $$
$$ ^{\ast }{\delta}^{15}{\mathrm{N}}_{\mathrm{foliage}}={f}_{\mathrm{N}\mathrm{O}3-}\times {\delta}^{15}{\mathrm{N}}_{\mathrm{N}\mathrm{O}3-}+{f}_{\mathrm{N}\mathrm{H}4+}\times {\delta}^{15}{\mathrm{N}}_{\mathrm{N}\mathrm{H}4+}+{f}_{\mathrm{DON}}\times {\delta}^{15}{\mathrm{N}}_{\mathrm{DON}} $$

In this study, the “Iso-Source” model was used to resolve this mathematically undetermined set of equations (Phillips and Gregg 2003; Houlton et al. 2007; Takebayashi et al. 2010; Zhang et al. 2018). This model iteratively generates source isotopic mixtures whose proportions (f) sum to 1. The model compares each calculation against a known mixture of δ15N foliage and retaining only those mixtures that satisfy the known value (within some mass-balance tolerance) as defined by a dataset of feasible solutions. Although this model can only generate feasible solutions (presented here as the average probability, the distribution of feasible solutions can be found in the supporting information), it nevertheless provides a systematic method to constrain the attribution of N sources in an undetermined system. In our case, the calculated mixtures reflected combinations of the δ15N of DON, ammonium, nitrate, and foliage. We applied a mass-balance tolerance of 0.5‰ to our calculations.

Statistical analysis

The differences in soil N concentrations and δ15N of different soil N forms among the four coniferous plantations were tested through one-way analysis of variance. P < 0.05 was considered to indicate a significant difference among coniferous plantations. All statistical analyses were conducted using SPSS 19.0 (SPSS Inc., Chicago, IL, USA)

Results

Soil N concentrations

DON was the dominant dissolved N form in both 0–10 cm and 10–20 cm mineral soil of the four coniferous plantations, with concentrations ranged from 23.4 to 34.7 mg N kg−1 in the 0–10 cm depth, and 14 to 18.3 mg N kg−1 in the 10–20 cm depth (Fig. 1). Ammonium and nitrate concentrations ranged from 9 to 16.1 mg N kg−1 and 4.2 to 5.9 mg N kg−1 in the 0–10 cm depth, and 5.1 to 9.4 mg N kg−1 and 2 to 3.4 mg N kg−1 in the 10–20 cm depth (Fig. 1). Soil ammonium concentrations differed significantly among four plantations in the 0–10 cm depth, with the highest ammonium concentrations in Picea koraiensis plantation (P < 0.05) (Fig. 1).

Fig. 1
figure1

Soil N concentration in the 0–10 cm and 10–20 cm mineral soil of the four studied forest plantations (means ± SE, n = 5). Concentration of ammonium and nitrate in mineral soil were reported in Zhou et al. (2020)

δ15N of different soil N forms and foliage

The δ15N of soil bulk N was 3.7 to 4.6‰ in the 0–10 cm depth, and 4.6 to 6.3‰ in the 10–20 cm depth (Fig. 2). After weighting by the N pool sizes for the two soil layers, the δ15N of soil DON ranged from 2.9 to 7.7‰ (Fig. 3). The δ15N of DON in Pinus sylvestris was significantly higher than the other three plantations (P < 0.05). There were no significant differences in the δ15N of soil ammonium and nitrate among the four plantations (P > 0.05), with ammonium from 2.5 to 5.8‰ and nitrate from −0.7 to 3.9‰ (Fig. 3). The δ15N of foliage ranged from −2.2 to 0.2‰ in the four plantations (Fig. 3), with the highest value for Pinus sylvestris and the lowest value for Pinus koraiensis.

Fig. 2
figure2

Soil δ15N values in the 0–10 cm and 10–20 cm mineral soil of the four studied forest plantations (means ± SE, n = 5)

Fig. 3
figure3

δ15N values of foliage and of soil DON, NH4+, and NO3 in 0–20 cm mineral soil (means ± SE, n = 5) in the four forest plantations. Different letters on bar of the same color indicate significant differences among four forest plantations in their δ15N values (one-way ANOVA, P < 0.05)

Soil N contribution for plant N uptake

Nitrate contributed 60 to 82% of total inorganic N uptake by four plantations based on the two end-member calculations (Fig. 4a). There was no significant difference of the nitrate contribution among the four plantations (P > 0.05) (Fig. 4a). When DON was incorporated as an available N source in our calculation, DON contributed 31%, 29%, 28%, and 24% to total N uptake by Larix olgensis, Picea koraiensis, Pinus koraiensis, and Pinus sylvestris, respectively, based on the three end-member calculations, whereas nitrate contributed 42 to 52% and ammonium contributed 19 to 29% to total N uptake for these four conifers (Fig. 5a). According to the results of the in situ labelling experiment, DON contributed 18 to 52% of total N uptake by the four conifers, nitrate contributed 34 to 53%, and ammonium contributed 11 to 35% (Fig. 5b).

Fig. 4
figure4

Contribution of NH4+ and NO3 to plant N uptake in the four forest plantations calculated by the two end-member (a) and from in situ labelling experiment in the previous study (b) (Zhou et al. 2020)

Fig. 5
figure5

Contribution of DON, NH4+, and NO3 to plant N uptake in the four forest plantations calculated by the three end-member (a), and calculated according to the results of in situ labelling experiment in the previous study (b, Methods and Materials, Zhou et al. 2020).

Discussion

Contribution of DON in comparison with DIN

Our results suggested that all four mature conifers could assimilate DON efficiently, with DON contributing 24 to 31% to total N uptake in the three end-member calculations and 18 to 52% in the in situ labelling experiment (Fig. 5). Consistent with previous studies using 15N natural abundance (Table 1), organic N plays a key role in plant N acquisition (Takebayashi et al. 2010; Zhang et al. 2018). For example, the study carried out on the Tibetan Plateau suggested that DON contributed 23 to 39% of plant N uptake across 20- to 70-year-old Picea asperata plantations (Zhang et al. 2018). Organic N uptake may allow conifers to circumvent microbial transformations to inorganic N. This effective competition with soil microbes for organic N will improve nutrient utilization efficiency of conifers (Harrison et al. 2007; Andersen et al. 2017), especially in relatively N-limited ecosystems.

Table 1 The proportional contribution of different N forms to plant N uptake in forest trees by using the 15N natural abundance method

Previous studies suggested that plant N uptake strategy may be related to the concentration of soil available N and that plants prefer to take up the most available N form in their rhizosphere (Russo et al. 2013; Mayor et al. 2014). Such uptake pattern could be an adaptive strategy for plants in nutrient competition (Andersen et al. 2017). In our study, DON was the dominant N form and accounted for almost 60% of dissolved soil N pools (Fig. 1). This might explain that mature conifers could take up a relatively large proportion of DON in this study, thereby reducing competition for the limited available N sources with microbes (Song et al. 2015). DON uptake by conifers can be energized by the proton gradient over the plasma membrane and facilitated by transport proteins (Näsholm et al. 2009; Svennerstam et al. 2011). These transporters can obtain amino acids from the soil solution, as well as from the roots (Jones et al. 2005). Previous studies have demonstrated that transporters in conifer root epidermis have broad affinity for many kinds of amino acids, which could contribute to the uptake of DON by conifers (Uscola et al. 2017). Furthermore, N uptake strategy of ectomycorrhizal plants is largely mediated by mycorrhizal fungi (Hobbie et al. 2000; Hobbie and Högberg 2012; Wang et al. 2020). The four conifers in our study are associated with ectomycorrhizal fungi, which can mobilize and take up organic N (Chalot et al. 2002; Näsholm et al. 2009). Overall, based on the 15N natural abundance method, the contribution of soil DON to nutrient uptake of mature conifers in forests of Northeast China deserves further attention.

Nitrate contribution in N uptake in comparison with the 15N labelling method

Nitrate was the important N source for the four conifers, with nitrate contributing 60 to 82% of total inorganic N uptake in the two end-member model (Fig. 4a) and 42 to 52% of total N uptake in the three end-member model (Fig. 5a). These results were consistent with the in situ labelling experiment that nitrate was preferentially taken up by conifers, accounting for 59 to 77% of total N uptake (Fig. 4b) (Zhou et al. 2020). Such uptake patterns differed from the results of hydroponic experiments in which conifers took up more ammonium than nitrate (Kronzucker et al. 1997; Socci and Templer 2011; Gruffman et al. 2014; Uscola et al. 2017), with ammonium contributing 55 to 98% of total N uptake (Zhou et al. 2020). However, the hydroponic method could not represent the uptake patterns of mature conifers in the field, since it ignores microbial competition for ammonium and adsorption of ammonium by soil minerals (Lavoie et al. 1992; Lucash et al. 2005; Fraterrigo et al. 2011). In addition, nitrate efflux is induced when plant roots are immersed directly in nitrate-labelled solutions (Socci and Templer 2011), resulting in underestimates of nitrate uptake by hydroponic methods (Wei et al. 2015).

The potential mechanisms for the efficient uptake and assimilation of nitrate are as follows: (1) Nitrate is usually more available for uptake in many ecosystems due to its high mobility (Johnson et al. 2000; Liu et al. 2017), while ammonium is preferentially assimilated by soil microbes and easily adsorbed by soil organic matter (Wang and Macko 2011; Epron et al. 2016). One study in a temperate grassland demonstrated that nitrate diffused through soil water more than one hundred times faster than ammonium (Owen and Jones 2001). Therefore, efficient assimilation of nitrate by conifers could avoid competition for ammonium with soil microbes and increase nutrient assimilation, despite the higher energy cost for nitrate assimilation than for ammonium assimilation in conifers (Kuzyakov et al. 2013), especially under low concentrations of soil available N (Zhu et al. 2019). (2) Nitrate could promote uptake of other nutrients, such as K+, Ca2+, and Mg2+, and thereby contribute to plant growth (Hoffmann et al. 2007). In contrast, ammonium assimilation and the accompanying equimolar H+ production could acidify the rhizosphere and consequently repress cation uptake (Britto and Kronzucker 2002). (3) Different active nitrate uptake systems may promote nitrate uptake by plant roots (Liu et al. 2014). Previous studies suggested that conifers may have low-affinity transport systems (LATS) and high-affinity transport systems (HATS) (Behl et al. 1988), which can contribute to efficient nitrate uptake by conifers at low and high soil nitrate concentrations.

Uncertainties of the 15N natural abundance method

In this study, we corrected the foliage δ15N by 5‰ in our calculation due to isotope fractionation during internal plant N allocation and ectomycorrhizal fungi transport (see “Methods and Materials”) (Hobbie and Colpaert 2003; Houlton et al. 2007; Zhang et al. 2018). However, we must admit that the isotopic fractionation effect may cause some uncertainties due to the different N status of different study coniferous plantations (Takebayashi et al. 2010; Zhang et al. 2018). To assess the uncertainties of conifer N uptake strategy, we tested the sensitivity of the two and three end-member calculations to a wide range of the isotope effect during plant N uptake from 4 to 12‰ (correction of 2–6‰ for the mixing model). We found that N uptake strategies of conifers were relatively sensitive to the isotope effect from 4 to 8‰ (i.e., correction of 2–4‰, Fig. 6). However, we calculated the 15N depletion of tree δ15N relative to soil inorganic N based on the previous in situ labelling experiment, with 15N depletion ranged from 4 to 6‰ (Zhou et al. 2020). Only marginal effects on the relative contribution of different N forms resulted when the isotope effect varied from 8 to 12‰ (correction of 4–6‰). Therefore, the relative contribution of different N forms calculated by the corrected isotope effect of 5‰ is robust in this study.

Fig. 6
figure6

Sensitivity analysis of the proportion of plant N derived from different N sources to the internal plant isotope effect by three and two end-member calculation. (a), (b), and (c) show the effect on the proportional contributions of different N sources to total N uptake by three end-member calculation, when considering combined isotope effect associated with N uptake, transportation and N assimilation (from 2‰ to 6‰); (d) and (e) show the proportional contributions of different N sources to total N uptake by two end-member calculation

We note that DON is a complex mixture of different N-containing substances. The δ15N of soil DON varies across the components of this mixture, and the DON uptake by different conifers of these components may vary as well (Yu et al. 2002; Andersson and Berggren 2005). This could lead to a difference in δ15N between the DON in the soil and the DON taken up by conifers. Therefore, we selected five conifers in each plantation to minimize these impacts. Furthermore, compared to the 15N labelling method, the 15N natural abundance method would be affected by environmental factors, such as the seasonal changes of δ15N of different soil N forms (Liu et al. 2014). Thus, multiple sampling times should be tested in the future to examine whether seasonal differences exist in the N uptake strategies of conifers.

Conclusions

Overall, efficient uptake of DON and nitrate by mature conifers in this study was consistent with the previous in situ experiment of these four conifers (Zhou et al. 2020). These results demonstrated that tree productivity and timber production in coniferous plantations should remain high, despite increasing proportions of nitrate in N deposition (Liu et al. 2013; Ackerman et al. 2018). However, the in situ labelling method was expensive to apply at field scales. Furthermore, it is difficult to study the uptake patterns of DON by mature conifers by the in situ labelling method. In contrast, the 15N natural abundance method was relatively convenient and practical (Zhang et al. 2018). Therefore, we suggest to use the 15N natural abundance method in the future to explore whether efficient utilization of nitrate is universal and generalizable across conifers.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

DON:

Dissolved organic N

δ15N:

Natural abundance of 15N

TDN:

Total dissolved N

LATS:

Low-affinity transport systems

HATS:

High-affinity transport systems

References

  1. Ackerman D, Millet DB, Chen X (2018) Global estimates of inorganic nitrogen deposition across four decades. Glob Biogeochem Cycle 33:100–107 https://doi.org/10.1029/2018GB005990

  2. Andersen KM, Mayor JR, Turner BL (2017) Plasticity in nitrogen uptake among plant species with contrasting nutrient acquisition strategies in a tropical forest. Ecology 98(5):1388–1398 https://doi.org/10.1002/ecy.1793

  3. Andersson P, Berggren D (2005) Amino acids, total organic and inorganic nitrogen in forest floor soil solution at low and high nitrogen input. Water Air Soil Pollut 162(1/4):369–384 https://doi.org/10.1007/s11270-005-7372-y

  4. Behl R, Tischner R, Raschke K (1988) Induction of a high-capacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism. Planta 176(2):235–240 https://doi.org/10.2307/23379355

  5. Boczulak SA, Hawkins BJ, Roy R (2014) Temperature effects on nitrogen form uptake by seedling roots of three contrasting conifers. Tree Physiol 34:513–523 https://doi.org/10.1093/treephys/tpu028

  6. Britto DT, Kronzucker HJ (2002) NH4+ toxicity in higher plants: a critical review. J Plant Physiol 159(6):567–584 https://doi.org/10.1078/01761610222260815

  7. Chalot M, Javelle A, Blaudez D et al (2002) An update on nutrient transport processes in ectomycorrhizas. Plant Soil 244(1):165–175 https://doi.org/10.1007/978-94-017-1284-2_16

  8. Craine JM, Elmore AJ, Aidar MPM et al (2009) Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability. New Phytol 183:980–992 https://doi.org/10.1111/j.1469-8137.2009.02917.x

  9. Dawson TE, Mambelli S, Plamboeck AH, Templer PH, Tu KP (2002) Stable isotopes in plant ecology. Ann Rev Ecol Syst 33:507–559 https://doi.org/10.1146/annurev.ecolsys.33.020602.095451

  10. Doyle A, Weintraub M, Schimel J (2004) Persulfate digestion and simultaneous colorimetric analysis of carbon and nitrogen in soil extracts. Soil Sci Soc Am J 68(2):669–676 https://doi.org/10.2136/sssaj2004.6690

  11. Epron D, Koutika L, Tchichelle SV, Bouillet J, Mareschal L (2016) Uptake of soil mineral nitrogen by Acacia mangium and Eucalyptus urophylla × grandis: no difference in N form preference. J Plant Nutr Soil Sci 179(6):726–732 https://doi.org/10.1002/jpln.201600284

  12. Fraterrigo JM, Strickland MS, Keiser AD, Bradford MA (2011) Nitrogen uptake and preference in a forest understory following invasion by an exotic grass. Oecologia 167(3):781–791 https://doi.org/10.1007/s00442-011-2030-0

  13. Gao L, Cui XY, Hill PW, Guo YF (2020) Uptake of various nitrogen forms by co-existing plant species in temperate and cold-temperate forests in northeast China. Appl Soil Ecol 147:103398 https://doi.org/10.1016/j.apsoil.2019.103398

  14. Gruffman L, Jämtgård S, Näsholm T (2014) Plant nitrogen status and co-occurrence of organic and inorganic nitrogen sources influence root uptake by Scots pine seedlings. Tree Physiol 34:1–9 https://doi.org/10.1093/treephys/tpt121

  15. Guo DL, Xia MX, Wei X, Chang WJ, Liu Y, Wang ZQ (2008) Anatomical traits associated with absorption and mycorrhizal colonization are linked to root branch order in twenty-three Chinese temperate tree species. New Phytol 180(3):673–683 https://doi.org/10.1111/j.1469-8137.2008.02573.x

  16. Harrison KA, Bol R, Bardgett RD (2007) Preferences for different nitrogen forms by coexisting plant species and soil microbes. Ecology 88:989–999 https://doi.org/10.1890/06-1018

  17. Hobbie EA, Colpaert JV (2003) Nitrogen availability and colonization by mycorrhizal fungi correlate with nitrogen isotope patterns in plants. New Phytol 157:115–126 https://doi.org/10.1046/j.1469-8137.2003.00657.x

  18. Hobbie EA, Högberg H (2012) Nitrogen isotopes link mycorrhizal fungi and plants to nitrogen dynamics. New Phytol 196(2):367–382 https://doi.org/10.1111/j.1469-8137.2012.04300.x

  19. Hobbie EA, Macko SA, Williams M (2000) Correlations between foliar delta 15N and nitrogen concentrations may indicate plant-mycorrhizal interactions. Oecologia 122(2):273–283 10.1007/PL00008856

  20. Hoffmann A, Milde S, Desel C et al (2007) N form-dependent growth retardation of Arabidopsis thaliana seedlings as revealed from physiological and microarray studies. J Plant Nutr Soil Sci 170(1):87–97 https://doi.org/10.1002/jpln.200625032

  21. Houlton BZ, Sigman DM, Schuur EAG, Hedin LO (2007) A climate-driven switch in plant nitrogen acquisition within tropical forest communities. Proc Natl Acad Sci USA 104(21):8745–8750 https://doi.org/10.1073/pnas.0609935104

  22. Johnson DW, Cheng W, Burke IC (2000) Biotic and abiotic nitrogen retention in a variety of forest soils. Soil Sci Soc Am J 64(4):1503–1514 https://doi.org/10.2136/sssaj2000.6441503x

  23. Jones DL, Shannon D, Junvee-Fortune T, Farrarc JF (2005) Plant capture of free amino acids is maximized under high soil amino acid concentrations. Soil Biol Biochem 37(1):179–181 https://doi.org/10.1016/j.soilbio.2004.07.021

  24. Knapp AN, Sigman DM, Lipschultz F (2005) N isotopic composition of dissolved organic nitrogen and nitrate at the Bermuda Atlantic time-series study site. Glob Biogeochem Cycle 19(1):1–15 https://doi.org/10.1029/2004GB002320

  25. Kronzucker HJ, Siddiqi MY, Glass ADM (1997) Conifer root discrimination against soil nitrate and the ecology of forest succession. Nature 385(6611):59–61 https://doi.org/10.1038/385059a0

  26. Kuzyakov Y, Xu X (2013) Competition between roots and microorganisms for nitrogen: mechanisms and ecological relevance. New Phytol 198(3):656–669 https://doi.org/10.1111/nph.12235

  27. Lavoie N, Vézina L, Margolis HA (1992) Absorption and assimilation of nitrate and ammonium ions by jack pine seedlings. Tree Physiol 11:171–183 https://doi.org/10.1093/treephys/11.2.171

  28. Li CC, Li QR, Qiao N, Xu XL, Li QK, Wang HM (2015) Inorganic and organic nitrogen uptake by nine dominant subtropical tree species. iForest 9:253–258 https://doi.org/10.3832/ifor1502-008

  29. Liu M, Li CC, Xu XL et al (2017) Organic and inorganic nitrogen uptake by 21 dominant tree species in temperate and tropical forests. Tree Physiol 37:1515–1526 https://doi.org/10.1093/treephys/tpx046

  30. Liu XJ, Zhang Y, Han WX et al (2013) Enhanced nitrogen deposition over China. Nature 494(7438):459–462 https://doi.org/10.1038/nature11917

  31. Liu XY, Koba K, Makabe A, Liu CQ (2014) Nitrate dynamics in natural plants: insights based on the concentration and natural isotope abundances of tissue nitrate. Front Plant Sci 5:1–14 https://doi.org/10.3389/fpls.2014.00355

  32. Lucash MS, Joslin JD, Yanai R (2005) Temporal variation in nutrient uptake capacity by intact roots of mature loblolly pine. Plant Soil 272(1):253–262 https://doi.org/10.1007/s11104-004-5296-8

  33. Mayor JR, Wright SJ, Schuur EAG, Brooks ME, Turner BL (2014) Stable nitrogen isotope patterns of trees and soils altered by long-term nitrogen and phosphorus addition to a lowland tropical rainforest. Biogeochemistry 119(1-3):293–306 https://doi.org/10.1007/s10533-014-9966-1

  34. McKane RB, Johnson LC, Shaver GR et al (2002) Resource-based niches provide a basis for plant species diversity and dominance in arctic tundra. Nature 415:68–71 https://doi.org/10.1038/415068a

  35. Näsholm T, Kielland K, Ganeteg U (2009) Uptake of organic nitrogen by plants. New Phytol 182(1):31–48 https://doi.org/10.1111/j.1469-8137.2008.02751.x

  36. Öhlund J, Näsholm T (2004) Regulation of organic and inorganic nitrogen uptake in Scots pine (Pinus sylvestris) seedlings. Tree Physiol 24:1397–1402 https://doi.org/10.1093/treephys/24.12.1397

  37. Orwin KH, Kirschbaum MUF, St John MG, Dickie IA (2011) Organic nutrient uptake by mycorrhizal fungi enhances ecosystem carbon storage: a model-based assessment. Ecol Lett 14(5):493–502 https://doi.org/10.1111/j.1461-0248.2011.01611.x

  38. Owen AG, Jones DL (2001) Competition for amino acids between wheat roots and rhizosphere microorganisms and the role of amino acids in plant N acquisition. Soil Biol Biochem 33:651–657 https://doi.org/10.1016/S0038-0717(00)00209-1

  39. Peri PL, Ladd B, Pepper DA, Bonser SP, Laffan SW, Amelung W (2012) Carbon (δ13C) and nitrogen (δ15N) stable isotope composition in plant and soil in Southern Patagonia’s native forests. Glob Change Biol 18(1):311–321 https://doi.org/10.1111/j.1365-2486.2011.02494.x

  40. Persson J, Gardeström P, Näsholm T (2006) Uptake, metabolism and distribution of organic and inorganic nitrogen sources by Pinus sylvestris. J Exp Bot 57(11):2651–2659 https://doi.org/10.1093/jxb/erl028

  41. Phillips DL, Gregg JW (2003) Source partitioning using stable isotopes: coping with too many sources. Oecologia 136(2):261–269 https://doi.org/10.1007/s00442-003-1218-3

  42. Russo SE, Kochsiek A, Olney J, Thompson L, Miller AE, Tan S (2013) Nitrogen uptake strategies of edaphically specialized Bornean tree species. Plant Ecol 214:1405–1416 https://doi.org/10.1007/s11258-013-0260-4

  43. Socci AM, Templer PH (2011) Temporal patterns of inorganic nitrogen uptake by mature sugar maple (Acer saccharum Marsh.) and red spruce (Picea rubens Sarg.) trees using two common approaches. Plant Ecol Divers 4(2-3):141–152 https://doi.org/10.1080/17550874.2011.624557

  44. Song MH, Zheng LL, Suding KN, Yin TF, Yu FH (2015) Plasticity in nitrogen form uptake and preference in response to long-term nitrogen fertilization. Plant Soil 394(1-2):215–224 https://doi.org/10.1007/s11104-015-2532-3

  45. Svennerstam H, Jämtgård S, Ahmad I et al (2011) Transporters in Arabidopsis roots mediating uptake of amino acids at naturally occurring concentrations. New Phytol 191(2):459–467 https://doi.org/10.1111/j.1469-8137.2011.03699.x

  46. Takebayashi Y, Koba K, Sasaki Y, Fang YT, Yoh M (2010) The natural abundance of 15N in plant and soil-available N indicates a shift of main plant N resources to NO3- from NH4+ along the N leaching gradient. Rapid Commun Mass Spectrom 24(7):1001–1008 https://doi.org/10.1002/rcm.4469

  47. Uscola M, Villar-Salvador P, Oliet J, Warren CR (2017) Root uptake of inorganic and organic N chemical forms in two coexisting Mediterranean forest trees. Plant Soil 415(1):387–392 https://doi.org/10.1007/s1110401731726

  48. Wang LX, Macko SA (2011) Constrained preferences in nitrogen uptake across plant species and environments. Plant Cell Environ 34(3):525–534 https://doi.org/10.1111/j.1365-3040.2010.02260.x

  49. Wang SS, Chen AQ, Xie K et al (2020) Functional analysis of the OsNPF4.5 nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants. Proc Natl Acad Sci USA 117(28):16649–16659 https://doi.org/10.1073/pnas.2000926117

  50. Wei LL, Chen CR, Yu S (2015) Uptake of organic nitrogen and preference for inorganic nitrogen by two Australian native Araucariaceae species. Plant Ecol Divers 8(2):259–264 https://doi.org/10.1080/17550874.2013.871656

  51. Yu Z, Zhang Q, Kraus TEC, Dahlgren RA, Anastasio C, Zasoski RJ (2002) Contribution of amino compounds to dissolved organic nitrogen in forest soils. Biogeochemistry 61:173–198 https://doi.org/10.1023/A:1020221528515

  52. Zhang SS, Fang YT, Xi D (2015) Adaptation of micro-diffusion method for the analysis of 15N natural abundance of ammonium in samples with small volume. Rapid Commun Mass Spectrom 29:1297–1306 https://doi.org/10.1002/rcm.7224

  53. Zhang ZL, Li N, Xiao J et al (2018) Changes in plant nitrogen acquisition strategies during the restoration of spruce plantations on the eastern Tibetan Plateau, China. Soil Biol Biochem 119:50–58 https://doi.org/10.1016/j.soilbio.2018.01.002

  54. Zhou XL, Wang A, Hobbie EA et al (2020) Mature conifers assimilate nitrate as efficiently as ammonium from soils in four forest plantations. New Phytol 229(6):3184–3194 https://doi.org/10.1111/nph.17110

  55. Zhu FF, Dai LM, Hobbie EA et al (2019) Uptake patterns of glycine, ammonium, and nitrate differ among four common tree species of Northeast China. Front Plant Sci 10:799 https://doi.org/10.3389/fpls.2019.00799

Download references

Acknowledgements

Thanks to Xiusen Yang and Xuan Li of the Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China, for their help during analyzing the isotopes of soil available N and foliage.

Funding

This research was funded by the National Key Research and Development Program of China (2016YFA0600802), Key Research Program of Frontier Sciences of Chinese Academy of Sciences (QYZDB-SSW-DQC002), K.C. Wong Education Foundation (GJTD-2018-07), Liaoning Vitalization Talents Program (XLYC1902016), the National Natural Science Foundation of China (41773094, 41811530305, 31901134, 41571455, and 31770498), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA23070103), and Scientific Research Foundation of the Educational Department of Liaoning Province (L201908).

Author information

Affiliations

Authors

Contributions

XZ, AW, YL, and YF conceived the study. XZ and AW performed field work. XZ, AW, YL, and YF performed analyses. All authors contributed to the writing and read and approved the final manuscript.

Corresponding authors

Correspondence to Yinghua Li or Yunting Fang.

Ethics declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

Supporting information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhou, X., Wang, A., Hobbie, E.A. et al. Nitrogen uptake strategies of mature conifers in Northeastern China, illustrated by the 15N natural abundance method. Ecol Process 10, 36 (2021). https://doi.org/10.1186/s13717-021-00306-4

Download citation

Keywords

  • Nitrogen uptake preference
  • Organic nitrogen
  • Inorganic nitrogen
  • Coniferous plantation
  • 15N natural abundance
  • Isotopic mixing model