Hypoxia Affects Nitrogen Uptake and Distribution in Young Poplar (Populus × canescens) Trees

Bin Liu; Heinz Rennenberg; Jürgen Kreuzwieser

doi:10.1371/journal.pone.0136579

Abstract

The present study with young poplar trees aimed at characterizing the effect of O₂ shortage in the soil on net uptake of NO₃^- and NH₄⁺ and the spatial distribution of the N taken up. Moreover, we assessed biomass increment as well as N status of the trees affected by O₂ deficiency. For this purpose, an experiment was conducted in which hydroponically grown young poplar trees were exposed to hypoxic and normoxic (control) conditions for 14 days. ¹⁵N-labelled NO₃^- and NH₄⁺ were used to elucidate N uptake and distribution of currently absorbed N and N allocation rates in the plants. Whereas shoot biomass was not affected by soil O₂ deficiency, it significantly reduced root biomass and, consequently, the root-to-shoot ratio. Uptake of NO₃^- but not of NH₄⁺ by the roots of the trees was severely impaired by hypoxia. As a consequence of reduced N uptake, the N content of all poplar tissues was significantly diminished. Under normoxic control conditions, the spatial distribution of currently absorbed N and N allocation rates differed depending on the N source. Whereas NO₃^- derived N was mainly transported to the younger parts of the shoot, particularly to the developing and young mature leaves, N derived from NH₄⁺ was preferentially allocated to older parts of the shoot, mainly to wood and bark. Soil O₂ deficiency enhanced this differential allocation pattern. From these results we assume that NO₃^- was assimilated in developing tissues and preferentially used to maintain growth and ensure plant survival under hypoxia, whereas NH₄⁺ based N was used for biosynthesis of storage proteins in bark and wood of the trees. Still, further studies are needed to understand the mechanistic basis as well as the eco-physiological advantages of such differential allocation patterns.

Citation: Liu B, Rennenberg H, Kreuzwieser J (2015) Hypoxia Affects Nitrogen Uptake and Distribution in Young Poplar (Populus × canescens) Trees. PLoS ONE 10(8): e0136579. https://doi.org/10.1371/journal.pone.0136579

Editor: Manuel Reigosa, University of Vigo, SPAIN

Received: March 27, 2015; Accepted: August 5, 2015; Published: August 26, 2015

Copyright: © 2015 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information file.

Funding: The article processing charge was funded by the German Research Foundation (DFG) and the Albert Ludwigs University Freiburg in the funding programme Open Access Publishing. Bin Liu was supported by scholarships from China Scholarship Council (CSC, Grant# 2010630072), Graduiertenförderung (LGFG) Baden-Württemberg and the University of Freiburg. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

As an important constituent of amino acids, proteins, nucleic acids, N-based osmo-protectants and defence compounds, nitrogen (N) is an essential major nutrient of plants. Important N compounds taken up by plant roots are the inorganic NO₃^- and NH₄⁺ [1] as well as organic N such as amino acids [2–4]. The concentrations of the different N compounds in forest soils vary considerably [5, 6] and highly depend on processes such as leaching or volatilization of N, but also on microbial processes using N compounds as substrates, including immobilisation, mineralisation, nitrification and denitrification [7]. Such processes are strongly influenced by environmental conditions [8]. For example, soil O₂ deficiency favours denitrification which leads to reduced abundance of NO₃^- but to increased NH₄⁺ concentrations in the soil [9, 10], whereas in O₂ rich soils nitrification dominates over denitrification leading to the formation of NO₃^- from NH₄⁺ [11]. It is well understood that N uptake by roots is strongly affected by the abundance of other N compounds, as, for example, reduced N such as NH₄⁺ or amino acids inhibit NO₃^- net uptake of coniferous and deciduous trees [4, 12–14].

Waterlogging and flooding are common environmental constrains leading to O₂ deficiency in soils. Whereas energy metabolism is not limited under normoxia, O₂ availability below 30 kPa (“critical O₂ pressure” [15]) limits respiratory ATP generation under hypoxia. In contrast, under anoxia the absence of O₂ allows only insignificant ATP generation by respiration [16]. Consequently, waterlogging and flooding can cause an energy crisis in the plant tissues affected [17, 18]. To maintain energy metabolism, hypoxic tissues switch from respiration to fermentative processes, mainly alcoholic fermentation [16, 19, 20]. However, fermentation is an energetically inefficient pathway because it yields only 2 molecules ATP per molecule glucose consumed as compared to 38 molecules ATP formed in mitochondrial respiration. Analysis of the plant transcriptome has revealed that under O₂ deficiency plants minimize energy consumption by slowing down ATP demanding processes including growth, biosynthesis of polymers and active transport processes [21–24]. As a major nutrient, N uptake comprises ca. 80% of all nutrients absorbed by roots from the soil [25] and, therefore, constitutes a strong energy sink [26]. Particularly, the active uptake of the quantitatively important NO₃^- which mechanistically occurs via proton symport, strongly depends on the ATP consuming maintenance of the proton gradient across the plasma membrane. In contrast, NH₄⁺ uptake is energetically favored, because it occurs thermodynamically “downhill” at concentrations above 200–500 μM. At lower concentrations, it is considered secondarily active, e.g. occurring through ATP-dependent NH₄⁺ pumps or via NH₄⁺ /H⁺ cotransport [18, 27, 28, 29]. Consequently, root uptake of NO₃^- is impaired by soil O₂ deprivation, whereas the energetically more advantageous NH₄⁺ uptake seems to be less affected [30, 31]. However, in such studies NO₃^- and NH₄⁺ were supplied individually as sole N source, and it is still unknown, how soil O₂ deficiency affects NO₃^- and NH₄⁺ uptake, if both nutrients are supplied in combination.

NO₃^- taken up by the roots is channelled into assimilatory NO₃^- reduction in the roots of many tree species, which is in contrast to herbaceous plants assimilating NO₃^- mainly in green tissues [32, 33]. Thus, in trees, reduced N, mainly as amino acids, is transported in the transpiration stream to the leaves, and is further distributed and plant-internally cycled [12, 34, 35]. Such cycling seems to be a tree specific feature, which ensures supply of reduced N to N demanding tissues [36]. Also in poplar, NO₃^- assimilation can occur in the roots; however, if NO₃^- reduction capacity of roots is exceeded because of high soil NO₃^- availability, the surplus NO₃^- is transported to the shoot and assimilated in the leaves [37, 38]. NO₃^- assimilation needs the sequential action of the enzymes NO₃^- reductase (NR, EC 1.6.6.1) forming NO₂^-, and NO₂^- reductase (NiR, EC 1.7.2.1) generating NH₄⁺ in an energy demanding manner [39]. NH₄⁺ is then used for the biosynthesis of organic N in form of amino acids by the glutamine synthetase/glutamine-oxoglutarate aminotransferase (GS/GOGAT) system (GS, EC 6.3.1.2/ Fd-ferredoxin-GOGAT, EC 1.4.7.1; NADH-GOGAT, EC 1.4.1.14) [40–41] and subsequent transamination reactions [42].

Soil O₂ deficiency not only affects plant N uptake but also N metabolism at the physiological and the transcriptomic level [23]. Consistently, altered concentrations of amino acids, proteins and N-containing pigments have been observed in response to flooding [30, 43] with consequences for major plant processes such as photosynthesis [44]. In contrast, effects of soil O₂ deficiency on plant-internal distribution of N is scarcely studied. Impacts of soil O₂ deprivation on root-to-shoot transport of N-compounds can be assumed due to the often strongly slowed-down transpiration stream following soil hypoxia [45]. Because of the strong energy dependence of NO₃^- uptake and assimilation as well as phloem transport of reduced N, impairment of these processes under O₂ depletion must be assumed. The present study was performed to test the hypotheses that (i) net uptake of N, particularly of NO₃^-, by roots of young Gray poplar trees is affected by soil O₂ deficiency leading to reduced biomass formation and total N contents in the trees, that (ii) the trees’ transpiration stream will be slowed down in response to soil O₂ shortage, which will (iii) cause reduced allocation of N from roots to the leaves. As NO₃^- assimilation will be strongly reduced under conditions of O₂ limitation, we hypothesize differential effects on the allocation of N derived from NO₃^- and NH₄⁺. To test these hypotheses, we elucidated the spatial distribution of the currently absorbed N as affected by soil O₂ deficiency. We exposed the roots of poplar trees to normoxic and hypoxic conditions, supplied them with ¹⁵N-labelled NO₃^- and NH₄⁺ and followed the allocation and distribution of ¹⁵N through the whole plant.

Materials and Methods

Plant material and growth conditions

The present experiments were performed with four months old Gray poplar (Populus x canescens clone INRA 717 1-B4) seedlings, which were micro-propagated as described earlier [46]. Four weeks old poplar cuttings cultivated in sterile culture tubes were transplanted to plastic pots (13 cm × 13 cm × 13 cm) containing sand (Glaser Trockensand GmbH, Malsch, Germany) treated with 0.15% fungicide solution (Proplant, Dr. Stählem GmbH, Stade, Germany) to minimize growth of pathogenic fungi. Plantlets were supplied regularly with distilled water; in addition, they were fertilized twice a week with 200 ml 25% modified Hoagland solution [47] consisting of 0.6 mM KNO₃, 1.3 mM Ca(NO₃)₂ × 4 H₂O, 0.3 mM MgSO₄, 1.5 mM MgCl₂, 0.25 mM KH₂PO₄, 2.3 μM MnCl₂ × 4 H₂O, 10 μM H₃BO₃, 0.08 μM CuCl₂ × 4 H₂O, 0.2 μM ZnCl₂, 0.2 μM Na₂MoO₂ × 4 H₂O, 0.04 μM CoCl₂ × 6 H₂O, 22.5 μM Na-EDTA, 22.5 μM FeCl₂ (pH 5.5). The plants were grown under long day condition (16h light/8 h dark) at a temperature of 22±5°C for 4 months in a greenhouse.

Experimental setup and protocol for introducing hypoxia

For experiments the seedlings were carefully taken out of the pots. After removing the sand from roots, each seedling was transferred into an amber glass bottle, which was filled with 1 L Hoagland nutrient solution; trees were adapted to the hydroponic environment for three days. During this time, the solutions were aerated with ambient air by means of air pumps (Schemel & Goetz GmbH & Co KG, Offenbach a. M., Germany). To avoid evaporation of water from the nutrient solution, all bottles were tightly sealed with parafilm (Bemis Company, Inc., Neenah, USA). Hypoxia was implemented for 14 days by stopping aeration. As a consequence, the O₂ concentrations in the nutrient solution dropped to constant levels of 0.007±0.006 mg L^-1, whereas it remained constantly between 7–8 mg L^-1 in the aerated solutions as indicated by O₂ determination with an O₂ microsensor (Microx TX2; PreSens, Regensburg, Germany).

Transpiration rates

Transpiration rates of the seedlings were calculated by weighing the water loss from the bottles containing the nutrient solution every two to three days until the 11^th day of soil O₂ deficiency.

¹⁵N labelling and plant harvest

To study NO₃^- and NH₄⁺ net uptake and the distribution of currently absorbed N, ¹⁵N-labelling experiments were performed with 48 seedlings whose total root systems were exposed to either normal or reduced O₂ availability for 14 days. For this purpose, the non-labelled solutions were completely removed from the bottles and replaced by nutrient solutions containing either ¹⁴NH₄Cl and K¹⁵NO₃ or ¹⁵NH₄Cl and K¹⁴NO₃ (n = 10–12) at final concentrations of 2.0 mM N (10%-atom ¹⁵N-abundance). Before adding these solutions, they were aerated (normoxia) or bubbled with N₂ gas, in order to maintain the O₂ concentrations in the bottles containing the trees. Natural ¹⁵N-abundances were used for correcting ¹⁵N labelling of each plant tissue. For this purpose, in parallel with the labelling experiment, trees exposed to normoxic or hypoxic conditions (8 trees per treatment) were supplied with non-labelled nutrient solutions. Two hours after exposure to the labelled nutrient solutions, poplar seedlings were harvested. For this purpose, each plant was carefully taken out from the bottle; the root part was immediately washed with tap water and then washed again with demineralized water; the whole seedlings were divided into four main sections: (1) the top 40 cm representing the developing part of the shoot, (2) the middle 40 cm section representing the younger mature part of the shoot, (3) the bottom section, ca. 50 cm in length, representing the older mature shoot section, and (4) the root section. Each shoot section was further divided into leaf, petiole, wood and bark, and the root section was further separated into coarse roots (>2 mm diameter) and fine roots (≤2 mm diameter). All plant parts were weighed and oven dried at 60°C until weight constancy. Dry samples were weighed and stored at room temperature until ¹⁵N analysis.

Analysis of total N and ¹⁵N contents

Total N contents and ¹⁵N-abundances in different plant tissues (fine and coarse roots; leaves, petioles, wood and bark from the top 40 cm, middle 40 cm and lowest 50 cm shoot sections) were analyzed by a C/N 2500 analyzer (CE Instruments, Milan, Italy) coupled to a mass spectrometer (IR-MS, Finnigan MAT GmbH, Bremen, Germany). All dry tissues were well powdered and homogenized by a ball mill (MM 400, Retsch GmbH, Haan, Germany). Depending on the tissue to be analyzed, aliquots of 2.0 to 6.0 mg were weighed into tin capsules (IVA Analysentechnik, Meerbusch, Germany) which were burned into gases in the element analyzer and further analyzed in the mass spectrometer. For the calculation of total N contents in different tissues, plants exposed to hypoxia and treated with ¹⁵NO₃^-/¹⁴NH₄⁺ (10–12 biological replicates) were combined with plants treated with ¹⁴NO₃^-/¹⁵NH₄⁺ (10–12 biological replicates) because exposure to these differently labelled N sources cannot influence the total N content; thus, for this approach 20–24 biological replicates were used.

Calculation of ¹⁵N distribution, rates of ¹⁵N allocation and ¹⁵NO₃^- /¹⁵NH₄⁺ uptake

The ¹⁵N allocation rates into different tissues were calculated with Eq (1), (1) where NAR is the NO₃^- and NH₄⁺ allocation rate (nmol g^-1 DW h^-1); Δ¹⁵N_tissue the difference of ¹⁵N abundance (% of total N) of different tissues from ¹⁵N-treated plant and non-labelled control plants (natural ¹⁵N abundance); [N] the total N concentration (g N g^-1 DW); DW_total the total dry weight (g); DW_tissue the tissue dry weight (g); Δt the incubation time (h); M (N) the molecular weight of ¹⁵N (15 g mol^-1). The calculation of total ¹⁵N per tissue was based on the specific ¹⁵N contents of the labelling solution and tissue biomass. Total ¹⁵N per plant was calculated by summing up the total ¹⁵N contents in all tissues.

NO₃^- or NH₄⁺ uptake rates were calculated from the total ¹⁵N accumulation in the plants during the incubation period and were based on fresh weight of fine roots. For the calculation of NO₃^- and NH₄⁺ uptake rates, eqs (2) and (3) were used, (2) (3) where in Eq (2) NUR is the specific NO₃^- or NH₄⁺ net uptake rate (nmol g^-1 FW h^-1); Δ¹⁵N_plant the difference of ¹⁵N abundance (% of total N) of whole plants from ¹⁵N-treated plant and non-labelled control plants (natural ¹⁵N abundance); [N] the total N concentration (g N g^-1 DW); DW_total the total dry weight (g); FW_fr the fresh weight of fine roots (g); Δt the incubation time (h); M (N) the molecular weight of ¹⁵N (15 g mol^-1). In Eq (3) ¹⁵N_plant is total ¹⁵N abundance (atom percentage) in the whole plants; ¹⁵N_leaf.n, ¹⁵N_petiole.n, ¹⁵N_wood.n and ¹⁵N_bark.n the ¹⁵N abundances (atom percentage) in the respective tissues from three different positions of the shoots, i.e. top 40 cm, middle 40 cm and lowest 50 cm; ¹⁵N_fr and ¹⁵N_cr are ¹⁵N abundances (atom percentage) in fine roots and coarse roots, respectively.

Statistical analysis

Data were tested for normality (Shapiro-Wilk test) and equality of variances. If required, we applied a logarithmic transformation (common logarithm) on the raw data. Significant differences between controls and hypoxia treated plants were determined using one-way analysis of variance (ANOVA) and Student’s t-test. When the normality test failed, the Kruskal-Wallis one-way ANOVA on ranks and the Mann-Whitney rank sum test were used instead. All statistical analyses were performed using Sigmaplot 11.0 (Systat Software GmbH, Erkrath, Germany).

Results

Growth parameters and transpiration

Poplar trees exposed to soil O₂ deficiency showed significantly decreased fine root biomass formation compared to trees grown at sufficient O₂ supply (Table 1). In contrast, most of the other plant organs did not show significant differences depending on soil O₂ availability. As a consequence, total biomass of poplar trees was the same under both treatments, but the root-to-shoot ratio decreased under soil O₂ deficiency (Fig 1). Rates of transpiration significantly decreased under hypoxia beginning from the 5^th day of the treatment (Fig 1).

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Table 1. Effect of soil O₂ deficiency (hypoxia) on biomass (g DW) of poplar plants.

https://doi.org/10.1371/journal.pone.0136579.t001

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Fig 1. Effect of soil O₂ deficiency on biomass accumulation and daily transpiration of young poplar trees.

Four months old, hydroponically grown poplar trees were exposed to either normoxic or hypoxic conditions. After 14 days of treatment the plants were harvested, oven dried and the dry weights determined. Root-to-shoot ratios were calculated for each plant. In addition, daily transpiration rates were determined. Data shown are means ± SD of 10–12 biological replicates per treatment. Statistically significant differences at p< 0.05 between hypoxic and normoxic plants were calculated by Student’s t-test and are shown by asterisk.

https://doi.org/10.1371/journal.pone.0136579.g001

Soil O₂ deficiency affects N content in plant tissues

We assessed total N contents in different above- and belowground parts of the poplar trees studied (Fig 2). Soil O₂ shortage significantly reduced the total N contents in all plant organs investigated. This effect was most pronounced in leaves and roots, where total N content decreased from 0.17±0.02 (normoxia) to 0.15±0.02 (hypoxia) g plant^-1 and 0.10±0.02 (normoxia) to 0.08±0.02 (hypoxia) g plant^-1, respectively (Fig 2A and 2B). The relative distribution of N, however, did not change due to O₂ deficiency. Leaves, for example, contained ca. 50% of total plant N independent on the treatment. Roots contained ca. 29% of total plant N, bark tissue ca. 13%, wood tissue ca.7% and the petioles ca. 2% of total plant N (Fig 2A and 2B). When expressed on a dry weight basis, hypoxia also resulted in significantly decreased N concentrations in all organs (Fig 2C). The N concentrations of the different above-ground plant organs depended on the position on the shoot (Table 2) with the uppermost plant parts consistently containing the highest N concentrations.

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Fig 2. Effects of hypoxia on the total N contents (A, B) and N concentrations (C) in organs of young poplar trees.

Trees were exposed to either normoxia (A) or hypoxia (B) for 14 days. After the treatment period, the plants were harvested, divided into the different parts, oven dried and after homogenization the total N contents (g organ^-1), relative portion of N (% of total N in plant) and concentrations (mmol g^-1 DW) determined. Data shown are means ± SD of 22–24 biological replicates. Statistically significant differences at p<0.05 between plants exposed to either hypoxia or normoxia were tested by Student’s t-test and are indicated by asterisks.

https://doi.org/10.1371/journal.pone.0136579.g002

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Table 2. N contents [mmol g^-1 DW] in poplar plants exposed to normoxia or hypoxia for 14 days.

https://doi.org/10.1371/journal.pone.0136579.t002

O₂ shortage affects ¹⁵NO₃^- but not ¹⁵NH₄⁺ net uptake

N uptake rates were determined after application of NH₄Cl in combination with KNO₃, where either the NH₄⁺ or the NO₃^- was labelled with ¹⁵N. At normal O₂ supply, NH₄⁺ uptake (778±293 nmol g^-1 FW h^-1) was about 3-times higher than NO₃^- uptake (259±75 nmol g^-1 FW h^-1) (Fig 3). Soil O₂ deficiency did not influence the uptake of NH₄⁺, however, NO₃^- uptake was significantly decreased (170±55 nmol g^-1 FW h^-1) under these conditions (Fig 3A). A very similar pattern with reduced NO₃^- but unaffected NH₄⁺ uptake was obtained if the N absorption at the whole plant level was calculated (Fig 3B).

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Fig 3. Effects of hypoxia on ¹⁵NO₃^- and ¹⁵NH₄⁺ uptake rate.

After a treatment period of 14 days, the poplar seedlings were supplied with either ¹⁴NH₄Cl and K¹⁵NO₃ or ¹⁵NH₄Cl and K¹⁴NO₃ at final N concentrations of 2.0 mM and incubated for 2 h. Trees were then harvested, ¹⁵N contents analyzed in dried tissues and N uptake rates calculated as described in materials and methods. Data shown are means ± SD of 10–12 biological replicates. The differences between hypoxic and normoxic control plants were calculated by LSD under ANOVA. Different lower case letters indicate statistical differences at p<0.05 between control and hypoxia treated poplar trees supplied with ¹⁵NO₃^- or ¹⁵NH₄⁺.

https://doi.org/10.1371/journal.pone.0136579.g003

Hypoxia alters the root-to-shoot distribution of N currently taken up

To investigate into which plant parts the currently absorbed N was distributed, the total ¹⁵N detected in roots and the shoot was assessed. The major parts of ¹⁵N derived from NO₃^- (i.e. ¹⁵NO₃^-N) or NH₄⁺ (i.e. ¹⁵NH₄⁺-N) taken up by the trees, was found in the roots (Fig 4A). Soil O₂ deficiency caused a significant decrease in root incorporated ¹⁵N. Under these conditions only 55±11% of the ¹⁵N absorbed as ¹⁵NO₃ remained in the roots compared to 73±3% under normal O₂ supply. Consequently, the portion of totally absorbed ¹⁵NO₃^-N which was transported from the roots to the shoot increased from 27±3% to 44±11%.

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Fig 4. Effect of hypoxia on the root—shoot distribution (A, B) and ¹⁵N allocation rates (C) in young poplar trees.

Young poplar trees were kept for 14 days under normoxic (A) or hypoxic (B) conditions and then supplied with ¹⁵NO₃^- or ¹⁵NH₄⁺ as described in legend of Fig 3. ¹⁵N contents were determined in all plant organs and data used to calculate the parameters shown. Data shown are means ± SD of 10–12 biological replicates. The differences between normoxia and hypoxia treated plants were tested by Student’s t-test at p< 0.05.

https://doi.org/10.1371/journal.pone.0136579.g004

O₂ shortage also reduced the portion of ¹⁵NH₄⁺-N which remained in the roots from 83±7% to 74±9% of the total ¹⁵N taken up (Fig 4B). Consequently, similar to NO₃, a significantly higher portion of the ¹⁵N absorbed by the roots was allocated to the shoot under these conditions. Irrespective of these changes, the allocation rates of ¹⁵NO₃^-N and ¹⁵NH₄⁺-N from roots to the shoot were unaffected by hypoxia (Fig 4C). Noteworthy, the allocation rates to the roots result from the balance of the rates of N net uptake and N transport from roots to shoot.

¹⁵NO₃^-N accumulates in developing leaves but ¹⁵NH₄⁺-N mainly in older sections of wood and bark

To study the distribution of currently absorbed ¹⁵N, all plant organs were separately analyzed for their ¹⁵N content. Under normal O₂ supply, the developing leaves and the younger mature leaves were strong sinks for ¹⁵NO₃^-N (Table 3). In the lowest, older parts of the plant, ¹⁵NO₃^-N was mainly detected in the wood. This pattern of distribution was enhanced under hypoxia, where the portions significantly increased from 3.3±1.1% to 6.3±3.3% (¹⁵N in youngest leaves) and from 11±2% to 22±13% (¹⁵N in oldest wood). Around 20% of the ¹⁵NO₃^-N taken up, accumulated in the coarse roots independent of the treatment. Fine roots of hypoxically treated plants contained ca. 36% less ¹⁵N than trees kept under normoxia.

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Table 3. Distribution of absorbed ¹⁵N (% of total ¹⁵N taken up per plant) in poplar plants kept under normoxia or hypoxia.

https://doi.org/10.1371/journal.pone.0136579.t003

In contrast to ¹⁵N derived from ¹⁵NO₃^-, where the young leaves were a major sink of ¹⁵N, the ¹⁵NH₄⁺-N was mainly found in wood, bark, petioles and leaves of the lowest parts of the trees (Table 3). This pattern was further enhanced if the trees were kept under conditions with reduced O₂ supply. The coarse roots contained somewhat less ¹⁵NH₄⁺-N (ca. 17% of the total ¹⁵NH₄⁺ taken up) than ¹⁵N from ¹⁵NO₃^-. With around 60% of the total ¹⁵NH₄⁺ absorbed by the plant, the fine roots accumulated the highest ¹⁵N content independent of the O₂ concentrations of the nutrient solution.

Effects of soil O₂ deficiency on N allocation rates

We calculated ¹⁵N allocation rates to the different plant parts of the poplar trees kept under different O₂ availability (Figs 5 and 6, S1 Fig). As expected from the ¹⁵N abundance in fine roots, the allocation of ¹⁵N to this organ dropped due to hypoxia by ca. 50% from 886±205 nmol g^-1 DW h^-1 to 411±154 nmol g^-1 DW h^-1 (Fig 5). Obviously, the trees allocated major portions of the ¹⁵N taken up to the developing and young mature leaves at rates of ca. 70–90 nmol g^-1 DW h^-1. These allocation rates were independent of the trees’ treatment. However, in contrast to the uppermost plant parts including leaves and bark, which were well supplied with ¹⁵NO₃^-N under soil O₂ deficiency, allocation rates to petioles and bark dropped in the middle and lowest part of the trees under these conditions.

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Fig 5. Effect of hypoxia on ¹⁵N allocation rates of ¹⁵N derived from ¹⁵NO₃^- in young poplar trees.

Poplar plants were treated as described in legend of Fig 3. ¹⁵N contents in all plant organs in different plant parts (top 40 cm, middle 40 cm, lowest 50 cm, fine and coarse roots) were determined and data used to calculate ¹⁵N allocation rates to these organs. The color codes indicate the magnitude of the allocation rates to the organs. Data shown are means ± SD of 10–12 biological replicates. Statistically significant differences between normoxic and hypoxic plants were tested by Student’s t-test and are indicated in S1 Fig.

https://doi.org/10.1371/journal.pone.0136579.g005

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Fig 6. Effect of hypoxia on ¹⁵N allocation rates of ¹⁵N derived from ¹⁵NH₄⁺ in young poplar trees.

Poplar plants were treated as described in legend of Fig 3. ¹⁵N contents in all plant organs in different plant parts (top 40 cm, middle 40 cm, lowest 50 cm, fine and coarse roots) were determined and data used to calculate ¹⁵N allocation rates to these organs. The color codes indicate the magnitude of the allocation rates to the organs. Data shown are means ± SD of 10–12 biological replicates. Statistically significant differences between plants exposed to normoxia or hypoxia were tested by Student’s t-test and are indicated in S1 Fig.

https://doi.org/10.1371/journal.pone.0136579.g006

The effects of O₂ shortage on the allocation rates of ¹⁵NH₄⁺-N clearly differed from that of ¹⁵NO₃^-. There was, for example, no difference between hypoxia and normoxia in the allocation rates into the fine roots (Fig 6), reflecting unaffected ¹⁵NH₄⁺ uptake and ¹⁵N allocation to the shoot under soil O₂ deficiency. Independent of the O₂ concentration of the nutrient solution, most of the ¹⁵NH₄⁺-N was transported to the older, i.e. lower, parts of the trees, which was in distinct contrast to the allocation of ¹⁵NO₃^-N. Hypoxia caused significantly lowered allocation rates of ¹⁵NH₄⁺-N to the young leaves (hypoxia: 11±7 nmol g^-1 DW h^-1; normoxia: 38±25 nmol g^-1 DW h^-1) and the petioles of the young mature part of the shoot (hypoxia: 39±26 nmol g^-1 DW h^-1; normoxia: 92±49 nmol g^-1 DW h^-1). The allocation rates to all other tissues were not significantly affected by soil O₂ shortage.

Discussion

Soil O₂ deprivation strongly impairs mitochondrial respiration causing a cellular energy crisis in the plant tissues affected [17]. As a consequence, ATP consuming processes such as nutrient uptake can be severely impaired [48–50]. In the present study, we focused on plant N metabolism and investigated N uptake and plant internal distribution of currently absorbed N as well as N allocation rates in poplar, a highly flood tolerant, riparian tree species.

Soil O₂ deprivation reduces N uptake by poplar roots

In accordance with previous studies on conifers and deciduous trees [6, 12, 51, 52], young poplar trees preferred NH₄⁺ over NO₃^- as N source. This might be a tree specific feature since herbaceous plants such as rice and maize took up NH₄⁺ and NO₃^- at similar rates [53]. Interestingly, in this study with crop plants a narrow part of the root (a few mm) directly behind the root tips also preferred NH₄⁺ over NO₃^-. In our study with poplar, NH₄⁺ was absorbed at ca. 3-times higher rates than NO₃^- under normal O₂ supply. In accordance to our hypothesis (i), this difference even increased under O₂ deprivation, because of significantly reduced NO₃^- uptake but unaffected NH₄⁺ absorption (Fig 3). Thus, although O₂ levels are not the causal explanation for the difference between NH₄⁺ and NO₃^- absorption under normal O₂ supply, reduced soil O₂ levels seem to exacerbate this situation. Importantly, reduced NO₃^- uptake was not only due (i) to lowered uptake rates on a root fresh weight basis but also (ii) to diminished root biomass (Table 1) enhancing the effects at the whole plant level (Fig 3). Such results are in good agreement with earlier studies indicating reduced NO₃^- absorption by roots of woody species [30, 31, 54]. In contrast, rice plants grown in a low O₂ root environment did not show reduced NO₃^- uptake most probably because under these conditions structural adaptation prevented O₂ loss from roots and ensured maintenance of an aerobic metabolism in the roots [55]. The preference of plants to different N sources depends on species and soil properties, for example, soil pH, temperature and abundances of different N forms [56]. The observed preferential absorption of NH₄⁺ over NO₃^- is often seen in tree species adapted to flood prone environments [30, 57, 58] and might be of ecological advantage, because the energy demand for NH₄⁺ uptake and assimilation is much lower than for NO₃^- use [59]. On the other hand, in riparian soils NH₄⁺ is more abundant than NO₃^- during flooding periods [9, 60]. This is, because under such conditions, NO₃^- can be (i) partially converted into NH₄⁺ by microorganisms, (ii) lost by leaching with flood water or (iii) volatilized and lost as gaseous N (N₂, N₂O) due to denitrification [7]. In consistence with the present work, very similar NO₃^- and NH₄⁺ uptake rates and effects of O₂ deficiency on N absorption were found in a former study with flooded poplar, where excised roots were supplied with NO₃^- or NH₄⁺ as the sole N source [31] and not in a combination of the two N sources as in the present study. In good agreement with diminished NO₃^- uptake, considerably reduced transcript levels of NO₃^- transporters were detected in hypoxia treated poplar roots [23].

Hypoxia affects total N content in poplar roots and shoot but biomass increment only of roots

In the present study, soil O₂ deficiency caused reduced fine root biomass formation whereas the biomass of the shoot and individual above-ground plant organs remained unaffected (Fig 1, Table 1). Decreased root biomass increment in trees in response to flooding has been observed frequently and was explained by impaired energy metabolism and reduced nutrient uptake [61–66]. Other studies also demonstrated reduced shoot growth which was related to impaired N status of the plants [67]. We assume that in our work the two weeks of soil O₂ deficiency of this highly flooding tolerant tree species was too short to cause shoot growth reduction. In our study, reduced fine root biomass occurred together with decreased NO₃^- uptake (as expressed on a fresh weight basis); thus, N uptake at the whole plant level considerably decreased under hypoxic conditions (Fig 3). This decline in N absorption was probably responsible for significantly lower N contents in all plant organs of hypoxically treated trees independent on their position on the shoot and regardless of the total amount of N per organ or the relative amount of N per dry weight (Fig 2, Table 2). These results are consistent with previous studies on several plant species including trees where flooding resulted in decreased amounts of total N in plant organs [62, 64, 68]. Such altered concentrations of important nutrients can cause strong nutritional imbalances within plants leading to growth retardation or injury [49]. Diminished leaf N content has been discussed as one reason for reduced rates of photosynthesis [44], which are often observed in flooded trees. Another reason for reduced gas exchange is the closure of stomata [45]; this was most probably also relevant in our study as suggested from the clearly reduced rates of transpiration in hypoxia treated trees compared to controls which supported our hypothesis (ii) (Fig 1).

The distribution pattern of N derived from NH₄⁺ and NO₃^- differs in poplar trees

Our results clearly indicated that the main portion of the N taken up remained in the roots, which might partially be due to the experimental procedure to harvest the plants directly after the labelling period. Still, this portion significantly decreased if the roots were exposed to soil O₂ shortage, i.e., higher portions of the N taken up were found in the shoot (Fig 4). To obtain a more detailed view of the fate of the NO₃^- and NH₄⁺ absorbed by the roots, we followed the ¹⁵N tracer in all plant parts in more detail. For the first time, our study demonstrated that the distribution pattern of N derived from NO₃^- and NH₄⁺ was different in poplar trees. The highest portion of the ¹⁵N derived from ¹⁵NO₃^- was found in the upper parts of the shoot, mainly in the developing and young mature leaves (Table 3). Similar preferential distribution of currently absorbed N to young mature and developing leaves was found in herbaceous plants [69]. Other studies with trees did not differentiate between different developmental stages of plant organs, but also demonstrated that the major portion of ¹⁵NO₃^- taken up by roots was allocated to the leaves [70, 71, 72]. Besides young leaves, wood of the lower parts of the stem was also a major sink of ¹⁵N derived from ¹⁵NO₃^-. Interestingly, in contrast to ¹⁵NO₃^-N, the greatest portion of ¹⁵NH₄⁺-N was detected in the lowest parts of the stems, namely in bark and wood and only small amounts in the developing leaves. Older leaves of the lower part of the shoot received significant amounts of the ¹⁵N taken up most probably for incorporation into storage proteins. Soil O₂ deprivation specifically enhanced this preferential distribution patterns of both ¹⁵NH₄⁺-N and ¹⁵NO₃^-N. Such findings seem to be new and similar observations have not been published before. We hypothesize that allocation of different N-forms occurs in a specific manner and speculate that a specific location of transporters mediating xylem unloading of N-compounds exist, which are influenced by the O₂ availability in the soil [73, 74].

N allocation rates are specifically altered by soil O₂ deprivation

Whereas total ¹⁵N contents in different plant parts indicate the relative distribution of the N absorbed by the plant (Table 3), N allocation rates provide better insight into the processes responsible for this distribution. In the present study we showed for the first time that the allocation rates of NO₃^- and NH₄⁺ from roots to the shoot were not affected by soil O₂ availability (Fig 4C). This is astonishing taken into account that the transpiration stream was severely slowed down under these conditions (Fig 1B). To maintain high N allocation rates between roots and the shoot, the xylem sap concentrations of N most probably strongly increased by soil O₂ shortage. These results suggest that xylem loading of N is not severely impaired by hypoxia and is widely independent of actual uptake rates of NO₃^- and NH₄⁺.

Highest allocation rates of ¹⁵NO₃^-N were observed to developing and young mature leaves (Fig 5, S1 Fig). We assume that most of the ¹⁵NO₃^-N was transported from root to the shoot in the form of NO₃^-. This assumption is indicated from the 10-fold higher in vivo NR activity and NR protein abundance in leaves than in roots of poplar trees [37, 38]. It is, therefore, generally assumed that young leaves of poplar are the main site of NO₃^- assimilation. In addition, we observed that a relatively high portion of the ¹⁵NO₃^-N accumulated in the youngest leaves (Table 2) supporting the latter assumption. Surprisingly, soil O₂ deprivation specifically influenced the allocation rates into individual plant parts. Despite reduced ¹⁵NO₃^- uptake by roots of hypoxia treated poplar trees, allocation rates of ¹⁵NO₃^-N into developing leaves remained unaffected. However, most organs from middle and lower parts of the shoot received less ¹⁵NO₃^-N under O₂ shortage. Only the allocation rates to the wood of these shoot sections were unaffected by hypoxia. As bark is considered an important N storage tissue for bark storage proteins (BSP) in poplar [75–77], reduced allocation rates into this tissue might indicate that storage of NO₃^-N in the bark was slowed down under O₂ deprivation. Such changed allocation pattern might be important to maintain N supply to physiologically active tissues in order to enable plant survival during hypoxia.

The allocation rates of ¹⁵NH₄⁺-N were completely different from that of ¹⁵NO₃^-N (Fig 6, S1 Fig). Highest ¹⁵NH₄⁺-N allocation rates were to the lower—older—plant parts, whereas allocation to the developing parts of the shoot was ca. 3-times slower. Such preferential translocation rates of different N sources to the older parts of the trees have not been described so far; it could be related to the biosynthesis of storage proteins in wood and bark tissue. The mechanism underlying such specific allocation is not clear and should be in the focus of future research. Specific allocation could be a consequence of xylem unloading of reduced N such as amino acids or ¹⁵NH₄⁺ [78], or of phloem transport of reduced N from leaves back to these tissues. From the present study it cannot be concluded, if ¹⁵NH₄⁺-N was transported in form of NH₄⁺ or as organic N, for example, as amino acids. Under normal O₂ supply, NH₄⁺ taken up is assimilated in the roots yielding glutamine and glutamate [79, 80]. It cannot be excluded that this energy demanding process is inhibited under O₂ deficiency as also suggested from gene expression data indicating reduced transcript abundance of glutamine synthetase (GS) and NADH-glutamine-oxoglutarate aminotransferase (NADH-GOGAT) in hypoxia treated poplar roots [23]. We therefore assume that the relative portion of NH₄⁺ which was transported from roots to the shoot increased under hypoxic conditions at the expense of amino acids.

Conclusion

Taken together, the observed N allocation patterns suggest that plant internal distribution of N is specific regarding the N source taken up by the roots. Moreover, it strongly depends on environmental conditions such as O₂ supply to the roots. In general, the observed allocation patterns of currently absorbed N derived from both ¹⁵NO₃^- and ¹⁵NH₄⁺ widely reflected the reduced N contents in the different plant organs under hypoxia. Changes in source-sink relations together with changes in xylem unloading processes might be responsible for such findings. Still, further research is needed to elucidate the underlying mechanisms for such compound specific N allocation patterns and the influence of soil O₂ deprivation on them.

Supporting Information

S1 Fig. Effect of hypoxia on ¹⁵N allocation rates of ¹⁵N derived from ¹⁵NO₃^- (A, B, C, D) and ¹⁵NH₄⁺ (E, F, G, H) in young poplar trees.

Poplar plants were treated as described in legend of Fig 3. ¹⁵N contents in all plant organs in different plant parts (A, E: top 40 cm, B, F: middle 40 cm, C, G: lowest 50 cm, D, H: fine and coarse roots) were determined and data used to calculate ¹⁵N allocation rates to these organs. The color codes indicate the magnitude of the allocation rates to the organs. Data shown are means ± SD of 10–12 biological replicates. Statistically significant differences at p<0.05 between normoxic and hypoxic plants were tested by Student’s t-test and are indicated by asterisks. TL, ML, LL: leaves of top, middle and lowest plant part; TP, MP, LP: petioles of top, middle, lowest part; TB, MB, LB: bark of top, middle, lowest part; TW, MW, LW: wood of top, middle lowest part. FR: fine roots, CR, coarse roots.

https://doi.org/10.1371/journal.pone.0136579.s001

(TIF)

Acknowledgments

We are grateful to Erika Fischer and Cornelia Blessing for technical support during the ¹⁵N tracer analysis.

Author Contributions

Conceived and designed the experiments: BL HR JK. Performed the experiments: BL. Analyzed the data: BL JK. Contributed reagents/materials/analysis tools: HR JK. Wrote the paper: BL HR JK.

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Figures

Abstract

Introduction

Materials and Methods

Plant material and growth conditions

Experimental setup and protocol for introducing hypoxia

Transpiration rates

15N labelling and plant harvest

Analysis of total N and 15N contents

Calculation of 15N distribution, rates of 15N allocation and 15NO3- /15NH4+ uptake

Statistical analysis

Results

Growth parameters and transpiration

Soil O2 deficiency affects N content in plant tissues

O2 shortage affects 15NO3- but not 15NH4+ net uptake

Hypoxia alters the root-to-shoot distribution of N currently taken up

15NO3-N accumulates in developing leaves but 15NH4+-N mainly in older sections of wood and bark

Effects of soil O2 deficiency on N allocation rates

Discussion

Soil O2 deprivation reduces N uptake by poplar roots

Hypoxia affects total N content in poplar roots and shoot but biomass increment only of roots

The distribution pattern of N derived from NH4+ and NO3- differs in poplar trees

N allocation rates are specifically altered by soil O2 deprivation

Conclusion

Supporting Information

S1 Fig. Effect of hypoxia on 15N allocation rates of 15N derived from 15NO3- (A, B, C, D) and 15NH4+ (E, F, G, H) in young poplar trees.

Acknowledgments

Author Contributions

References

¹⁵N labelling and plant harvest

Analysis of total N and ¹⁵N contents

Calculation of ¹⁵N distribution, rates of ¹⁵N allocation and ¹⁵NO₃^- /¹⁵NH₄⁺ uptake

Soil O₂ deficiency affects N content in plant tissues

O₂ shortage affects ¹⁵NO₃^- but not ¹⁵NH₄⁺ net uptake

¹⁵NO₃^-N accumulates in developing leaves but ¹⁵NH₄⁺-N mainly in older sections of wood and bark

Effects of soil O₂ deficiency on N allocation rates

Soil O₂ deprivation reduces N uptake by poplar roots

The distribution pattern of N derived from NH₄⁺ and NO₃^- differs in poplar trees

N allocation rates are specifically altered by soil O₂ deprivation

S1 Fig. Effect of hypoxia on ¹⁵N allocation rates of ¹⁵N derived from ¹⁵NO₃^- (A, B, C, D) and ¹⁵NH₄⁺ (E, F, G, H) in young poplar trees.