Characteristics of NH4+ and NO3− Fluxes in Taxodium Roots under Different Nitrogen Treatments

To understand the characteristics of net NH4+ and NO3− fluxes and their relation with net H+ fluxes in Taxodium, net fluxes of NH4+, NO3− and H+ were detected by a scanning ion-selective electrode technique under different forms of fixed nitrogen (N) and experimental conditions. The results showed that higher net NH4+ and NO3− fluxes occurred at 2.1–3.0 mm from the root apex in T. ascendens and T. distichum. Compared to NH4+ or NO3− alone, more stable net NH4+ and NO3− fluxes were found under NH4NO3 supply conditions, of which net NH4+ flux was promoted at least 1.71 times by NO3−, whereas net NO3− flux was reduced more than 81.66% by NH4+ in all plants, which indicated that NH4+ is preferred by Taxodium plants. T. ascendens and T. mucronatum had the largest net NH4+ and total N influxes when NH4+:NO3− was 3:1. 15N Atom% and activities of N assimilation enzymes were improved by single N fertilization in the roots of T. distichum. In most cases, net H+ fluxes were tightly correlated with net NH4+ and NO3− fluxes. Thus, both N forms and proportions could affect N uptake of Taxodium. These findings could provide useful guidance for N management for better productivity of Taxodium plants.


Introduction
Nitrogen (N) plays a significant role in plant growth and development since it is a crucial component of plants' chlorophylls, nucleic acids, proteins, and secondary metabolites [1]. Ammonium (NH 4 + ) and nitrate (NO 3 − ) are two primary forms of inorganic N absorbed and used by plants, and their fluxes in roots are varied with the distance from the apex. Spatial variability in the fluxes of NH 4 + and NO 3 − has been explored along the roots in some herbaceous and woody plants [2][3][4][5]. For instance, the maximal net NH 4 + influx happened at the root apex in rice (Oryza sativa L.) [2] and Populus simonii [5], whereas the highest net NH 4 + influx appeared at 5 mm, 10 mm, and 5-20 mm from the root apex in lodgepole pine (Pinus contorta) [6], Populus popularis [7] and Douglas-fir (Pseudotsuga menziesii) [6], respectively. In the case of NO 3 − , previous studies observed that the highest net NO 3 − flux occurred at 0-10 mm in P. contorta [6], and at 15 mm from the apex in P. simonii [5] and P. popularis [7]. In rice, net NO 3 − influx increased to a maximum at 21 mm from the apex and then gradually declined [2]. Obviously, different plant species have distinct patterns of NH 4 + and NO 3 − flux rates along the fine roots. Apart from the spatial variation along the roots, NH 4 + and NO 3 − fluxes are also affected by environmental factors such as N levels. A previous study in tea (Camellia sinensis) roots demonstrated increased net influxes of NH 4 + and NO 3 − when the solution concentration increased from 0.2 mM to 1.2 mM under KNO 3 and NH 4 Cl [1]. However, research on Picea glauca revealed a converse result in most cases; the roots presented net NH 4 + and NO 3 − influxes in 50 µM with net effluxes in 1500 µM solutions [8]. When roots were treated in 10, 100, and 1000 µM NH 4 NO 3 solutions, net NH 4 + influxes increased gradually in P. popularis but decreased by degrees in Populus alba × Populus glandulosa [4]. In contrast, the hybrid presented higher net NO 3 − influxes than P. popularis in most cases [4]. This phenomenon revealed that the N concentrations in soils have prominent effects on the uptake of NH 4 + and NO 3 − , and they are significantly related to the plant species. Additionally, previous studies revealed that interactions between NH 4 + and NO 3 − exist on fluxes of both ions [7,9]. The interactions between NH 4 + and NO 3 − are complicated among plants [10], and the underlying mechanisms remain unclear [7]. It is documented that the presence of NH 4 + and NO 3 − negatively affect the uptake of each other, but NH 4 + is preferred in C. sinensis [1]. However, net NH 4 + influx was induced by the simultaneous provision of NO 3 − , and net NO 3 − influx was inhibited in the presence of NH 4 + in roots of P. popularis and Populus asperata [7,11]. Moreover, a previous study on Douglas-fir and lodgepole pine showed that net NH 4 + uptake remained unchanged in the presence or absence of NO 3 − [6]. Overall, interactions between NH 4 + and NO 3 − and their preferences may result in changes of NH 4 + and NO 3 − fluxes under different proportions of NH 4 + and NO 3 − supply. Nonetheless, little information is available on the fluxes of NH 4 + and NO 3 − in plant roots under fluctuating proportions of both inorganic N forms.
On the other hand, fluxes of NH 4 + and NO 3 − are correlated with the plasma membrane PM-H + -ATPase activity that extrudes H + from the cytosol to the outside at the expense of adenosine triphosphate (ATP) [12]. Previous research found that NO 3 − is transported across the plasma membrane via NO 3 − /H + symporters with the involvement of PM-H + -ATPase [13]. The concentration of NH 4 + can increase the activity of PM-H + -ATPase [14]. Furthermore, the expression of genes encoding PM-H + -ATPase was positively associated with fluxes of NH 4 + and NO 3 − [8]. The significant correlations between NH 4 + , NO 3 − fluxes, and H + uptake rate have been observed in many plants [12,[15][16][17].
Taxodium species including T. ascendens, T. distichum, and T. mucronatum have been introduced from southeastern America to many countries owing to their economic and ecological benefits [18]. For instance, they can be used as woody bioenergy crops [19]. Taxodium oil showed adequate bioassay for insecticidal activity [20]. Compounds isolated from the bark can exhibit cytotoxic substances, thus treating against cancer cells [21]. Moreover, Taxodium plants have been selected as suitable species for afforestation in many challenging areas [22,23]. Although N is crucial for Taxodium growth and development, less information is available on the fluxes of NH 4 + and NO 3 − as well as their correlation with H + flux in fine roots. In this study, a non-invasive micro-electrodes technique was employed to investigate NH 4 + , NO 3 − and H + fluxes in fine roots of T. ascendens, T. distichum, and T. mucronatum under different N forms and their proportions. Our objectives were (i) to determine the distance from the root apex of Taxodium plants where there are greater net NH 4 + and NO 3 − fluxes; (ii) to illustrate the characteristics of NH 4 + , NO 3 − and H + fluxes and their interactions under different N forms and proportions.

Plant Cultivation
Semi-lignified cuttings (10 cm in length, 0.3 cm in diameter) of T. ascendens, T. distichum, and T. mucronatum were selected. After being soaked in 3‰ 3-indoleacetic acid (IAA) solution for 2 min, they were repotted into a pot containing 1:1 volume of peat: perlite in a ventilated greenhouse at the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (35 • [24]. All nutrient solutions were continuously aerated with an air pump, and each solution was refreshed every other day. After 16 d, plants were used to explore 15 N Atom% and enzymatic activities.

Experimental Design
To determine the positions along the root where the maximal influxes of NH 4 + and NO 3 − occur, a preliminary experiment was carried out at 14 positions, in turns, 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.5, 3.0, 5.0, 8.0, 15.0 and 30.0 mm away from the root apex. The measuring solution was 0.5 mM MES (2-(N-Morpholino) ethanesulfonic acid hydrate buffer.), pH 6.0, to which either 1.0 mM NH 4 Cl for NH 4 + or 1.0 mM KNO 3 for NO 3 − was added. After that, the position where the greater net uptake of NH 4 + and NO 3 − occurred was detected to carry out the following experiments.
To investigate the net fluxes of NH 4 + and NO 3 − under different N forms and proportions, the measuring solutions were designed as (1)  To explore the biomass, 15 N Atom% and enzymatic activities under single N fertilization, 24 seedlings with similar performance (ca. 15 cm in height) were selected and divided into three groups (8 plants in each group). Three N treatments: 0 mM 15 NH 4 Cl and K 15 NO 3 (serving as control, CK), 1 mM 15 NH 4 Cl and 1 mM K 15 NO 3 in 1/4 modified Hoagland's nutrient solution [24] were applied. Dicyandiamide (7 µM, C 2 H 4 N 4 ) was added into the nutrient solution to inhibit nitrification [19]. After 3 d, 4 plants from each treatment were harvested and used for measurements of 15 N Atom%, and the remaining 4 plants in each treatment were used for enzymatic activities.

Measurement of NH 4 + , NO 3 − and H + Fluxes
To understand the real-time NH 4 + , NO 3 − and H + uptake by the fine roots under different treatments, ions flux alterations on the root surface were measured by using a noninvasive micro-test technology (NMT) system (youngerusa.com; xuyue.net) (Figure 1a). used to explore 15 N Atom% and enzymatic activities.

Experimental Design
To determine the positions along the root where the maximal influxes of NH4 + and NO3 − occur, a preliminary experiment was carried out at 14 positions, in turns, 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.5, 3.0, 5.0, 8.0, 15.0 and 30.0 mm away from the root apex. The measuring solution was 0.5 mM MES (2-(N-Morpholino) ethanesulfonic acid hydrate buffer.), pH 6.0, to which either 1.0 mM NH4Cl for NH4 + or 1.0 mM KNO3 for NO3 − was added. After that, the position where the greater net uptake of NH4 + and NO3 − occurred was detected to carry out the following experiments.
To explore the biomass, 15 N Atom% and enzymatic activities under single N fertilization, 24 seedlings with similar performance (ca. 15 cm in height) were selected and divided into three groups (8 plants in each group). Three N treatments: 0 mM 15 NH4Cl and K 15 NO3 (serving as control, CK), 1 mM 15 NH4Cl and 1 mM K 15 NO3 in 1/4 modified Hoagland's nutrient solution [24] were applied. Dicyandiamide (7 μM, C2H4N4) was added into the nutrient solution to inhibit nitrification [19]. After 3 d, 4 plants from each treatment were harvested and used for measurements of 15 N Atom%, and the remaining 4 plants in each treatment were used for enzymatic activities.

Measurement of NH4 + , NO3 − and H + Fluxes
To understand the real-time NH4 + , NO3 − and H + uptake by the fine roots under different treatments, ions flux alterations on the root surface were measured by using a noninvasive micro-test technology (NMT) system (youngerusa.com; xuyue.net) (Figure 1a). The measurement procedures were described by Zhao et al. [5]. Firstly, ion-selective microelectrodes designed with 2-4 μm apertures were manufactured and silanized. Secondly, for the NH4 + electrode, in sequence, 100 mM NH4Cl was used as a backfilling solution, followed by an NH4 + selective liquid ion exchange cocktail (#09879, Sigma, St. Louis, MI, USA). Similarly, for the NO3 − electrode, 10 mM KNO3 was used as the backfilling solution, followed by a NO3 − selective liquid ion exchange cocktail (#72549, Sigma). For the The measurement procedures were described by Zhao et al. [5]. Firstly, ion-selective microelectrodes designed with 2-4 µm apertures were manufactured and silanized. Secondly, for the NH 4 + electrode, in sequence, 100 mM NH 4 Cl was used as a backfilling solution, followed by an NH 4 + selective liquid ion exchange cocktail (#09879, Sigma, St. Louis, MI, USA). Similarly, for the NO 3 − electrode, 10 mM KNO 3 was used as the backfilling solution, followed by a NO 3 − selective liquid ion exchange cocktail (#72549, Sigma). For the H + electrode, 15 mM NaCl and 40 mM KH 2 PO 4 were used as backfilling solution, followed by an H + selective liquid ion exchange cocktail (#95293, Sigma). Prior to the flux measurements, the microelectrodes were calibrated. For NH 4 + calibration, 0.05/0.5 mM NH 4 Cl in addition to other compounds (0.5 mM MES, pH 6.4/5.4) were used in the measuring solution; for NO 3 − calibration, 0.05/0.5 mM KNO 3 in addition to the compounds (0.5 mM MES, pH 6.4/5.4) were used in the measuring solution; for H + calibration, pH 6.4/5.4 in addition to 0.5 mM MES were used in the measuring solution. Only electrodes with Nernstian slopes higher than 55 mV per tenfold concentration difference were used.
After that, fine white roots, 15-35 mm from the apex, were selected. They were fixed at the bottom of the petri dish filled with 10-20 mL measuring solutions for 20 min. After being equilibrated, the samples were transferred to another petri dish containing 5 mL fresh solution and then were put under the microscope. The tips of the microelectrodes were aligned and kept 30 µm away from the target point, which is a specific distance from the apex of the root. Net fluxes of NH 4 + /NO 3 − were recorded at each measurement point for 5 min. Not only eight biological repetitions (eight fine roots from four plants) but also 50 measurement time points in each repetition were considered.

Determination of 15 N Uptake and Enzyme Activities
The roots were harvested and rinsed three times in distilled water. The 15 N Atom% ( 15 N AT%) and the amount of plant N derived from 15 N-labeled fertilizer (Ndff %) were detected [25]. Activities

Statistical Analysis
In order to determine the NH 4 + , NO 3 − and H + fluxes along the root tip, imFluxes V2.0 (xuyue.net) was used to obtain the data at each measuring point. The positive values represent net influxes, and the net negative values represent net effluxes. To analyze data for the ion fluxes, biomass, 15 N AT% and enzyme activities, one-way ANOVA (Duncan's multiple range tests at 5% level) was performed with the SPSS 25.0 (Statistical Product and Service Solutions, IBM, New York, NY, USA). GraphPad Prism version 9.1 was used to draw figures.

Net Fluxes of NH 4 + and NO 3 − along the Root Tip
Net fluxes of NH 4 + and NO 3 − were determined along the root tip up to 30.0 mm from the apex, and their fluxes were widely varied at different locations (Figure 2a

Net Fluxes of NH4 + and NO3 − under Different N Forms
As NH4Cl and KNO3 were added separately, the NH4 + and NO3 − fluxes fluctu widely for all tested plants at 2.5 mm from the root apex during a 5-min period (Figur Both NH4 + and NO3 − fluxes of T. distichum and T. mucronatum showed a tendency tow net influx. T. ascendens, however, tended to show net efflux of NH4 + and net influx of N (Figure 3a,d,g). When supplied with mixed N (NH4NO3), stable fluxes of NH4 + and N were observed, and distinctly, NH4 + fluxes were much greater than NO3 − fluxes in all odium plants (Figure 3b,e,h). Compared to 1 mM NH4Cl, average net fluxes of NH4 + w stimulated by 688%, 171%, and 762% under 1 mM NH4NO3 in roots of T. ascenden distichum, and T. mucronatum, respectively (Figure 3c,f,i). Thus, the increase of NH4 + fl was as follows: T. mucronatum > T. ascendens > T. distichum (Figure 3c

Net Fluxes of NH 4 + and NO 3 − under Different N Forms
As NH 4 Cl and KNO 3 were added separately, the NH 4 + and NO 3 − fluxes fluctuated widely for all tested plants at 2.5 mm from the root apex during a 5-min period ( Figure 3). Both NH 4 + and NO 3 − fluxes of T. distichum and T. mucronatum showed a tendency towards net influx. T. ascendens, however, tended to show net efflux of NH 4 + and net influx of NO 3 − (Figure 3a,d,g). When supplied with mixed N (NH 4 NO 3 ), stable fluxes of NH 4 + and NO 3 − were observed, and distinctly, NH 4 + fluxes were much greater than NO 3 − fluxes in all Taxodium plants (Figure 3b,e,h). Compared to 1 mM NH 4 Cl, average net fluxes of NH 4 + were stimulated by 688%, 171%, and 762% under 1 mM NH 4 NO 3 in roots of T. ascendens, T. distichum, and T. mucronatum, respectively (Figure 3c,f,i). Thus, the increase of NH 4 + fluxes was as follows: T. mucronatum > T. ascendens > T. distichum (Figure 3c,f,i). The same order was observed for the decreases in net NO 3 − fluxes, which were decreased by 314%, 220%, and 81.66% under 1 mM NH 4 NO 3 compared with that under 1 mM KNO 3 , respectively (Figure 3c,f,i).

Net NH4 + , NO3 − and H + Fluxes under Different N Concentrations
Except for T. ascendens exposed to 1.0 mM NH4Cl, all three species showed a tendency for net NH4 + influx when fed with 0.1 or 1.0 mM NH4Cl (Figure 4a). Additionally, the net influx of NH4 + in T. ascendens was significantly greater (p < 0.05) than those in the other two species under 0.1 mM NH4Cl (Figure 4a). Compared to 0.1 mM NH4Cl, net influx of NH4 + was promoted by 2. 40 (Figure 4b). Moreover, the fluxes of NO3 − were significantly lower (p < 0.05) in 1.0 mM than in 0.1 mM KNO3 in T. mucronatum (Figure 4b).
At the same time, net H + fluxes were determined in this study (Figure 4c). Here, we found that H + presented net effluxes under all treatments except for T. ascendens under 1.0 mM KNO3, T. distichum under 0.1 mM NH4Cl, and T. mucronatum under 1.0 mM NH4Cl (Figure 4c). Other than T. distichum exposed to 0.1 mM NH4Cl, the change tendency of net H + fluxes was similar to the variations of net NH4 + and NO3 − fluxes when the solution concentration was increased from 0.1 mM to 1.0 mM (Figure 4c).

Net NH 4 + , NO 3 − and H + Fluxes under Different N Concentrations
Except for T. ascendens exposed to 1.0 mM NH 4 Cl, all three species showed a tendency for net NH 4 + influx when fed with 0.1 or 1.0 mM NH 4 Cl ( Figure 4a). Additionally, the net influx of NH 4 + in T. ascendens was significantly greater (p < 0.05) than those in the other two species under 0.1 mM NH 4 Cl (Figure 4a). Compared to 0.1 mM NH 4 Cl, net influx of NH 4 + was promoted by 2. 40 (Figure 4b). Moreover, the fluxes of NO 3 − were significantly lower (p < 0.05) in 1.0 mM than in 0.1 mM KNO 3 in T. mucronatum (Figure 4b).
At the same time, net H + fluxes were determined in this study (Figure 4c). Here, we found that H + presented net effluxes under all treatments except for T. ascendens under 1.0 mM KNO 3 , T. distichum under 0.1 mM NH 4 Cl, and T. mucronatum under 1.0 mM NH 4 Cl (Figure 4c). Other than T. distichum exposed to 0.1 mM NH 4 Cl, the change tendency of net H + fluxes was similar to the variations of net NH 4 + and NO 3 − fluxes when the solution concentration was increased from 0.1 mM to 1.0 mM (Figure 4c).
Compared with the net NH4 + influx, the net flux of NO3 − was much lower, ranging from −100.53 to 84.85 pmol cm −2 s −1 under different proportions of NH4Cl and KNO3 (Figure 5b). It is surprising that the net influx of NO3 − observed under the 1:3 solution was replaced by net efflux when the proportion changed to 1:1 and 3:1 in T. ascendens ( Figure  5b). T. distichum, however, presented a totally converse trend whereby the net flux of NO3 − significantly (p < 0.05) increased by 1.68 times when the NH4 + proportion was raised from 1:1 to 3:1 (Figure 5b). In the case of T. mucronatum, there was net efflux under 1:3 and 1:1 and net influx under 3:1 (Figure 5b).
The trend of TN fluxes ranging from 114.23 to 1500.48 pmol cm −2 s −1 was similar to the fluxes of NH4 + (Figure 5c), and the highest net influx of TN was observed for T. mucronatum and T. ascendens when the proportion of NH4Cl: KNO3 was 3:1 (Figure 5c). Among the three species, T. distichum displayed the lowest net NH4 + , NO3 − and TN fluxes in all measuring solutions (Figure 5c).
In addition, the net H + fluxes were determined in this study (Figure 5d). All the treatments showed net H + influx when the two forms of N were provided together (Figure 5d).
Compared with the net NH 4 + influx, the net flux of NO 3 − was much lower, ranging from −100.53 to 84.85 pmol cm −2 s −1 under different proportions of NH 4 Cl and KNO 3 (Figure 5b). It is surprising that the net influx of NO 3 − observed under the 1:3 solution was replaced by net efflux when the proportion changed to 1:1 and 3:1 in T. ascendens (Figure 5b). T. distichum, however, presented a totally converse trend whereby the net flux of NO 3 − significantly (p < 0.05) increased by 1.68 times when the NH 4 + proportion was raised from 1:1 to 3:1 (Figure 5b). In the case of T. mucronatum, there was net efflux under 1:3 and 1:1 and net influx under 3:1 (Figure 5b).
The trend of TN fluxes ranging from 114.23 to 1500.48 pmol cm −2 s −1 was similar to the fluxes of NH 4 + (Figure 5c), and the highest net influx of TN was observed for T. mucronatum and T. ascendens when the proportion of NH 4 Cl: KNO 3 was 3:1 (Figure 5c). Among the three species, T. distichum displayed the lowest net NH 4 + , NO 3 − and TN fluxes in all measuring solutions (Figure 5c).
In addition, the net H + fluxes were determined in this study (Figure 5d). All the treatments showed net H + influx when the two forms of N were provided together (Figure 5d).

15 N AT%, Ndff% and Enzyme Activities in the Roots of T. distichum
Compared with CK, 15 N AT% was elevated by 70.27% or 29.73% in 1 mM 15 NH 4 +treated or 1 mM 15 NO 3 − -treated T.distichum roots (Table 1). Similar results were also observed in Ndff% (Table 1). Compared to CK, however, no significant difference was found in the root biomass of T. distichum supplied with 1 mM 15 NH 4 + or 15 NO 3 − during the 3 d experiment period (Table 1).  15 NO 3 − fertilization also has positive impacts on the activities of N assimilation enzymes (Table 2). When compared with CK, NR, NiR, GS, GDH and GOGAT activities were enhanced by 50.63%, 33.74%, 39.40%, 48.25% and 59.36%, respectively in 1 mM 15 NH 4 +supplied T. distichum roots (Table 2). Similarly, the activities of NR and NiR were increased by 221.03% and 11.93%, respectively in 1 mM 15 NO 3 − -fed T. distichum roots (Table 2).

Spatial Variability of Net NH 4 + and NO 3 − Fluxes along the Fine Roots
Fine roots consist of four distinct regions, including root cap, meristematic, elongation, and maturation zones, characterized by different anatomical and functional features [7]. These anatomical and functional diversities could bring about distinct absorbing abilities for NH 4 + and NO 3 − in different root zones [26][27][28]. Spatial variability of net NH 4 + and/or NO 3 − flux has been observed in fine roots of various plant species [29]. For example, maximal net NH 4 + influx occurred at the root apex in rice [2] and P. simonii [5], and at 5 mm, 10 mm, and 5-20 mm from the root apex in P. contorta [6] and P. popularis [7], and Douglas-fir [6] respectively. Such spatial variation of net NH 4 + and NO 3 − influxes along the root axis was also observed in our research. The largest net influxes of NH 4 + and NO 3 − were detected at m from the apex of T. ascendens and T. distichum, which belongs to the elongation zone. Such differences are possibly because of cytosolic concentrations of NH 4 + and NO 3 − in the elongation zone being lower than the thresholds needed for N assimilation to support the fast growth [30,31]. Similar results were observed in studies of Arabidopsis, where larger net NH 4 + fluxes were shown in the elongation zones [28,32]. Moreover, Phyllostachys edulis showed relatively higher net influxes of NH 4 + and NO 3 − at 2-5 mm from the root apex [30]. The net NH 4 + or NO 3 − fluxes were found to be higher in segment I (0-35 mm) than segment II (35-70 mm) in Populus × canescens [31]. In addition, we found that T. distichum had the greatest NH 4 + and NO 3 − uptake rates among the three Taxodium species.

Net NH 4 + and NO 3 − Fluxes under Single N Treatments
Generally, environmental N levels have a significant impact on the NH 4 + and NO 3 − fluxes of fine roots [4]. For instance, gradual increases in the fluxes of NH 4 + and/or NO 3 − were determined when supplied N was elevated in P. popularis and P. alba × P. glandulosa [4], and C. sinensis [1]. However, the opposite results were observed in P. glauca [8], wheat [24], and corn (Zea mays L.) [33]. Although an increasing N supply is likely to enhance N uptake in most cases, the provision of just NH 4 + could lead to soil acidification [34]. In most cases, to maintain ion homeostasis, roots release H + while absorbing NH 4 + , decreasing pH in the growth medium [35,36]. Eventually, this may lead to physiological and morphological disturbance of plants and then bring about toxicity and low production [37]. For example, acidification can significantly induce aluminum absorption, which is harmful to the development of plants [38]. In contrast, after absorption of NO 3 − , OH − could be released, contributing to the increase of pH [39]. Thus, a balanced supply of NH 4 + and NO 3 − is expected to improve the N uptake of plants and the soil environment.

Net NH 4 + and NO 3 − Fluxes under Mixed N Treatments
Many studies had demonstrated that the uptake of NH 4 + and NO 3 − was affected by each other when both N forms were provided [1,7]. In this study, the presence of NO 3 − stimulated the uptake of NH 4 + , whereas the net fluxes of NO 3 − were inhibited by NH 4 + in Taxodium plants. Similar results were found in the roots of corn, tea, wheat, rice and Brassica campestris [1,2,9,24,33], which indicated that NH 4 + and NO 3 − might interact with each other under coexistence N forms. These results might be related to cytosolic NH 4 + /NO 3 − thresholds [30]. In detail, more NH 4 + may be required for plant development when NO 3 − was provided, while pre-existing NH 4 + may reduce the thresholds of NO 3 − in the plant [1]. Considering that a higher net NH 4 + influx than NO 3 − was observed, it can be concluded that Taxodium plants show a preference for NH 4 + . It is noted that when NH 4 Cl or KNO 3 was solely supplied, the fluxes of NH 4 + or NO 3 − in the three species were erratic. However, stable net NH 4 + and NO 3 − fluxes were observed when NH 4 + and NO 3 − were both present in the solution, indicating a better balance in the mixed solution. In addition, this interesting phenomenon was reported in the study of C. sinensi, which might be the result of the competition between NH 4 + and NO 3 − , and the underlying mechanism needs to be further studied [1].
Because of the greater N uptake in mixed treatments than in single N conditions, strong net uptake of NH 4 + in fine roots of Taxodium species was expected to occur when NH 4 + and NO 3 − were supplied in different proportions [37]. In the case of tea, the maximum net NH 4 + influx was observed when NH 4 + :NO 3 − was 1:1, and the highest net NO 3 − influx occurred when NH 4 + :NO 3 − was 1.2:1 [1]. In blueberry (Vaccinium corymbosum L.), the mRNA levels of ammonium transporter 3 (VcAMT3) involved in NH 4 + uptake as well as nitrate transporter 1.5 (VcNRT1.5) and VcNRT2 involved in NO 3 − uptake was highest when the NH 4 + :NO 3 − ratio was 2:1 [40]. The highest growth rate, which is positively correlated with N uptake, of T. aestivum L., Brachiaria brizantha, and Pseudostellaria heterophylla was found when the NH 4 + and NO 3 − were supplied equivalently [1,24,41,42]. In this study, the best uptake rates of N were found when NH 4 + :NO 3 − was 3:1, 1:3, and 3:1 for T. ascendens, T. distichum, and T. mucronatum, respectively, which could provide an applicable proportion of NH 4 + and NO 3 − when producing special N fertilizer for the productivity of Taxodium plants. Additionally, the optimal equilibrium between NH 4 + and NO 3 − supply largely differed between the three Taxodium species, implying that the induction of N transport systems require distinct NH 4 + and NO 3 − ratios among these plants. In the present study, we found that with the change in the proportion of NH 4 + : NO 3 − (total N concentration: 2 mM), the NH 4 + influxes were improved more than NO 3 − . This observation indicates a preference for NH 4 + over NO 3 − , which is in good agreement with our previous outcomes. In most plant species, NH 4 + is first absorbed into cells and then directly converted to amino acids, whereas cytosolic NO 3 − is assimilated at a higher energy cost. It is reduced to NO 2 − with the help of nitrate reductase (NR) and is further converted into NH 4 + in plastids by nitrite reductase (NiR), which requires more energy than NH 4 + for both transportation and further reduction [43]. On the other hand, the flux discrepancies between NH 4 + and NO 3 − might result from the lower activity of NO 3 − transport systems affected by NH 4 + , which reduces the expression of the NO 3 − -related genes [1]. In blueberry plants, the expression of AMTs and NRTs was largely affected by the different ratios of NH 4 + : NO 3 − [40]. A previous study has indicated that different AMTs determined the uptake of NH 4 + to a certain extent, which was mediated by the external concentration [44]. Furthermore, various AMTs and NRTs have different substrate affinities appropriate to different N concentrations [5,8,10]. Therefore, the complicated fluxes of NH 4 + and NO 3 − when supplied at different proportions might be related to the distinct energetic and biochemical characteristics of uptake and assimilation pathway between NH 4 + and NO 3 − in plant roots [4,5,24].

Net NH 4 + and NO 3 − Fluxes Associated with H +
In this study, the alteration of H + fluxes was tightly associated with the variation in NH 4 + or NO 3 − . Previous studies revealed that H + fluxes might be correlated with the transport of NH 4 + and NO 3 − , since NH 4 + is transported into root cells through a symporter (co-transport with H + ) and/or a uniporter, and NO 3 − is co-transported with H + via a symporter into the cytosol [12][13][14]. Additionally, by maintaining a proton gradient, plasma membrane PM-H + -ATPase facilitates transport by pumping H + into the apoplast during the uptake of NH 4 + or NO 3 − in some parts of the roots [4, 43,45]. The activities of PM-H + -ATPase are determined by the transcript levels of corresponding mRNAs [4]. Although inconsistent results of H + fluxes under different N treatments were observed in the present study, H + still plays an essential role in plant uptake of NH 4 + and NO 3 − . Similar to a previous study in fine roots of P. popularis [7], our data indicated a tendency for net H + uptake when two forms of N were supplied simultaneously. Intriguingly, fluxes of H + fluctuated under both single N sources. Through our findings, we suspect that there may be an interaction between net H + flux and net NH 4 + /NO 3 − flux in roots of Taxodium species. Similar results were observed by Garnett et al. [15]. The specifics of the proposed interaction remain unclear. It is challenging to find out the specific mechanism underlying the correlation between H + and NH 4 + /NO 3 − in Taxodium roots.

Conclusions
In summary, spatial variability of NH 4 + and NO 3 − fluxes was observed along fine roots of Taxodium plants, and T. ascendens and T. distichum had higher fluxes of NH 4 + and NO 3 − at 2.1-3.0 mm from the root apex. In most cases, net fluxes of NH 4 + and NO 3 − increased with the elevated single N levels. NH 4 + and NO 3 − affected each other when they were both supplied, and Taxodium plants preferred NH 4 + . Higher net N influxes were found when NH 4 + and NO 3 − were simultaneously supplied than sole N treatments, especially in T. ascendens and T. mucronatum at 3:1 of NH 4 + :NO 3 − . Additionally, H + fluxes were tightly correlated with net NH 4 + and NO 3 − fluxes. These findings are valuable for understanding the characteristics of NH 4 + and NO 3 − fluxes in the fine roots of Taxodium plants in the context of single and various ratios of N supply, and could provide a scientific basis for N management for silvicultural practice and better productivity of Taxodium plants.

Institutional Review Board Statement:
The study did not require ethical approval, for studies not involving humans or animals.