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

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.


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 NO 3 and NH 4 + [1] as well as organic N such as amino acids [2][3][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 O 2 deficiency favours denitrification which leads to reduced abundance of NO 3 but to increased NH 4 + concentrations in the soil [9,10], whereas in O 2 rich soils nitrification dominates over denitrification leading to the formation of NO 3 from NH 4 + [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 NH 4 + or amino acids inhibit NO 3 net uptake of coniferous and deciduous trees [4,[12][13][14].
Waterlogging and flooding are common environmental constrains leading to O 2 deficiency in soils. Whereas energy metabolism is not limited under normoxia, O 2 availability below 30 kPa ("critical O 2 pressure" [15]) limits respiratory ATP generation under hypoxia. In contrast, under anoxia the absence of O 2 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 O 2 deficiency plants minimize energy consumption by slowing down ATP demanding processes including growth, biosynthesis of polymers and active transport processes [21][22][23][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 NO 3 which mechanistically occurs via proton symport, strongly depends on the ATP consuming maintenance of the proton gradient across the plasma membrane. In contrast, NH 4 + 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 NH 4 + pumps or via NH 4 + /H + cotransport [18,27,28,29]. Consequently, root uptake of NO 3 is impaired by soil O 2 deprivation, whereas the energetically more advantageous NH 4 + uptake seems to be less affected [30,31]. However, in such studies NO 3 and NH 4 + were supplied individually as sole N source, and it is still unknown, how soil O 2 deficiency affects NO 3 and NH 4 + uptake, if both nutrients are supplied in combination.
NO 3 taken up by the roots is channelled into assimilatory NO 3 reduction in the roots of many tree species, which is in contrast to herbaceous plants assimilating NO 3 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, NO 3 assimilation can occur in the roots; however, if NO 3 reduction capacity of roots is exceeded because of high soil NO 3 availability, the surplus NO 3 is transported to the shoot and assimilated in the leaves [37,38]. NO 3 - [42]. Soil O 2 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 O 2 deficiency on plant-internal distribution of N is scarcely studied. Impacts of soil O 2 deprivation on root-to-shoot transport of N-compounds can be assumed due to the often strongly sloweddown transpiration stream following soil hypoxia [45]. Because of the strong energy dependence of NO 3 uptake and assimilation as well as phloem transport of reduced N, impairment of these processes under O 2 depletion must be assumed. The present study was performed to test the hypotheses that (i) net uptake of N, particularly of NO 3 -, by roots of young Gray poplar trees is affected by soil O 2 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 O 2 shortage, which will (iii) cause reduced allocation of N from roots to the leaves. As NO 3 assimilation will be strongly reduced under conditions of O 2 limitation, we hypothesize differential effects on the allocation of N derived from NO 3 and NH 4 + . To test these hypotheses, we elucidated the spatial distribution of the currently absorbed N as affected by soil O 2 deficiency. We exposed the roots of poplar trees to normoxic and hypoxic conditions, supplied them with 15 N-labelled NO 3 and NH 4 + and followed the allocation and distribution of 15 N through the whole plant.

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 (

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.

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 O 2 deficiency.

N labelling and plant harvest
To study NO 3 and NH 4 + net uptake and the distribution of currently absorbed N, 15 N-labelling experiments were performed with 48 seedlings whose total root systems were exposed to either normal or reduced O 2 availability for 14 days. For this purpose, the non-labelled solutions were completely removed from the bottles and replaced by nutrient solutions containing either 14 NH 4 Cl and K 15 NO 3 or 15 NH 4 Cl and K 14 NO 3 (n = 10-12) at final concentrations of 2.0 mM N (10%-atom 15 N-abundance). Before adding these solutions, they were aerated (normoxia) or bubbled with N 2 gas, in order to maintain the O 2 concentrations in the bottles containing the trees. Natural 15 N-abundances were used for correcting 15 N 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 15 N analysis.

Analysis of total N and 15 N contents
Total N contents and 15 N-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 15 where NAR is the NO 3 and NH 4 + allocation rate (nmol g -1 DW h -1 ); Δ 15 N tissue the difference of 15 N abundance (% of total N) of different tissues from 15 N-treated plant and non-labelled control plants (natural 15 N 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 15 N (15 g mol -1 ). The calculation of total 15 N per tissue was based on the specific 15 N contents of the labelling solution and tissue biomass. Total 15 N per plant was calculated by summing up the total 15 N contents in all tissues. NO 3 or NH 4 + uptake rates were calculated from the total 15 N accumulation in the plants during the incubation period and were based on fresh weight of fine roots. For the calculation of NO 3 and NH 4 + uptake rates, eqs (2) and (3) were used, where in Eq (2) NUR is the specific NO 3 or NH 4 + net uptake rate (nmol g -1 FW h -1 ); Δ 15 N plant the difference of 15 N abundance (% of total N) of whole plants from 15 N-treated plant and non-labelled control plants (natural 15 N 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 15 N (15 g mol -1 ). In Eq (3)

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).

Growth parameters and transpiration
Poplar trees exposed to soil O 2 deficiency showed significantly decreased fine root biomass formation compared to trees grown at sufficient O 2 supply (Table 1). In contrast, most of the other plant organs did not show significant differences depending on soil O 2 availability. As a consequence, total biomass of poplar trees was the same under both treatments, but the rootto-shoot ratio decreased under soil O 2 deficiency (Fig 1). Rates of transpiration significantly decreased under hypoxia beginning from the 5 th day of the treatment (Fig 1).

Soil O 2 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 O 2 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 O 2 deficiency. Leaves, for example, contained ca. 50% of total plant N independent on the treatment. Roots contained ca. 29% of total plant N, bark  (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. 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.  3). Soil O 2 deficiency did not influence the uptake of NH 4 + , however, NO 3 uptake was significantly decreased (170±55 nmol g -1 FW h -1 ) under these conditions (Fig 3A). A very similar pattern with reduced NO 3 but unaffected NH 4 + uptake was obtained if the N absorption at the whole plant level was calculated (Fig 3B).

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 15 N detected in roots and the shoot was assessed. The major parts of 15 N derived from NO 3 -(i.e.  15 N 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 15   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. To study the distribution of currently absorbed 15 N, all plant organs were separately analyzed for their 15 (Figs 5 and 6, S1 Fig). As expected from the 15 N abundance in fine roots, the allocation of 15 N 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 15 N 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 15 NO 3 -N under soil O 2 deficiency, allocation rates to petioles and bark dropped in the middle and lowest part of the trees under these conditions. The effects of O 2 shortage on the allocation rates of 15 NH 4 + -N clearly differed from that of 15 NO 3 -. There was, for example, no difference between hypoxia and normoxia in the allocation rates into the fine roots (Fig 6), reflecting unaffected 15

Discussion
Soil O 2 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][49][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 O 2 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 NH 4 + over NO 3 as N source. This might be a tree specific feature since herbaceous plants such as rice and maize took up NH 4 + and NO 3 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 NH 4 + over NO 3 -. In our study with poplar, NH 4 + was absorbed at ca. 3-times higher rates than NO 3 under normal O 2 supply. In accordance to our hypothesis (i), this difference even increased under O 2 deprivation, because of significantly reduced NO 3 uptake but unaffected NH 4 + absorption (Fig 3). Thus, although O 2 levels are not the causal explanation for the difference between NH 4 + and NO 3 absorption under normal O 2 supply, reduced soil O 2 levels seem to exacerbate this situation. Importantly, reduced NO 3 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 NO 3 absorption by roots of woody species [30,31,54]. In contrast, rice plants grown in a low O 2 root environment did not show reduced NO 3 uptake most probably because under these conditions structural adaptation prevented O 2 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 NH 4 + over NO 3 is often seen in tree species adapted to flood prone  15 N 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. doi:10.1371/journal.pone.0136579.g006 environments [30,57,58] and might be of ecological advantage, because the energy demand for NH 4 + uptake and assimilation is much lower than for NO 3 use [59]. On the other hand, in riparian soils NH 4 + is more abundant than NO 3 during flooding periods [9,60]. This is, because under such conditions, NO 3 can be (i) partially converted into NH 4 + by microorganisms, (ii) lost by leaching with flood water or (iii) volatilized and lost as gaseous N (N 2 , N 2 O) due to denitrification [7]. In consistence with the present work, very similar NO 3 and NH 4 + uptake rates and effects of O 2 deficiency on N absorption were found in a former study with flooded poplar, where excised roots were supplied with NO 3 or NH 4 + 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 NO 3 uptake, considerably reduced transcript levels of NO 3 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 O 2 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][62][63][64][65][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 O 2 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 NO 3 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 NH 4 + and NO 3 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 O 2 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 NO 3 and NH 4 + absorbed by the roots, we followed the 15 N tracer in all plant parts in more detail. For the first time, our study demonstrated that the distribution pattern of N derived from NO 3 and NH 4 + was different in poplar trees. The highest portion of the 15 N derived from 15 NO 3 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 15 NO 3 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 15  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 O 2 availability in the soil [73,74].

N allocation rates are specifically altered by soil O 2 deprivation
Whereas total 15 N 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 NO 3 and NH 4 + from roots to the shoot were not affected by soil O 2 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 O 2 shortage. These results suggest that xylem loading of N is not severely impaired by hypoxia and is widely independent of actual uptake rates of NO 3 and NH 4 + .
Highest allocation rates of 15 NO 3 -N were observed to developing and young mature leaves ( Fig 5, S1 Fig). We assume that most of the 15 NO 3 -N was transported from root to the shoot in the form of NO 3 -. 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 NO 3 assimilation. In addition, we observed that a relatively high portion of the 15 NO 3 -N accumulated in the youngest leaves (  [79,80]. It cannot be excluded that this energy demanding process is inhibited under O 2 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 NH 4 + 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 O 2 supply to the roots. In general, the observed allocation patterns of currently absorbed N derived from both 15 NO 3 and 15 NH 4 + 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 O 2 deprivation on them.
Supporting Information