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

Abstract

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.

1. 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 (NH₄⁺) and nitrate (NO₃⁻) 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 NH₄⁺ and NO₃⁻ has been explored along the roots in some herbaceous and woody plants [2,3,4,5]. For instance, the maximal net NH₄⁺ influx happened at the root apex in rice (Oryza sativa L.) [2] and Populus simonii [5], whereas the highest net NH₄⁺ 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 NO₃⁻, previous studies observed that the highest net NO₃⁻ 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 NO₃⁻ influx increased to a maximum at 21 mm from the apex and then gradually declined [2]. Obviously, different plant species have distinct patterns of NH₄⁺ and NO₃⁻ flux rates along the fine roots.

Apart from the spatial variation along the roots, NH₄⁺ and NO₃⁻ fluxes are also affected by environmental factors such as N levels. A previous study in tea (Camellia sinensis) roots demonstrated increased net influxes of NH₄⁺ and NO₃⁻ when the solution concentration increased from 0.2 mM to 1.2 mM under KNO₃ and NH₄Cl [1]. However, research on Picea glauca revealed a converse result in most cases; the roots presented net NH₄⁺ and NO₃⁻ influxes in 50 μM with net effluxes in 1500 μM solutions [8]. When roots were treated in 10, 100, and 1000 μM NH₄NO₃ solutions, net NH₄⁺ influxes increased gradually in P. popularis but decreased by degrees in Populus alba × Populus glandulosa [4]. In contrast, the hybrid presented higher net NO₃⁻ influxes than P. popularis in most cases [4]. This phenomenon revealed that the N concentrations in soils have prominent effects on the uptake of NH₄⁺ and NO₃⁻, and they are significantly related to the plant species. Additionally, previous studies revealed that interactions between NH₄⁺ and NO₃⁻ exist on fluxes of both ions [7,9]. The interactions between NH₄⁺ and NO₃⁻ are complicated among plants [10], and the underlying mechanisms remain unclear [7]. It is documented that the presence of NH₄⁺ and NO₃⁻ negatively affect the uptake of each other, but NH₄⁺ is preferred in C. sinensis [1]. However, net NH₄⁺ influx was induced by the simultaneous provision of NO₃⁻, and net NO₃⁻ influx was inhibited in the presence of NH₄⁺ in roots of P. popularis and Populus asperata [7,11]. Moreover, a previous study on Douglas-fir and lodgepole pine showed that net NH₄⁺ uptake remained unchanged in the presence or absence of NO₃⁻ [6]. Overall, interactions between NH₄⁺ and NO₃⁻ and their preferences may result in changes of NH₄⁺ and NO₃⁻ fluxes under different proportions of NH₄⁺ and NO₃⁻ supply. Nonetheless, little information is available on the fluxes of NH₄⁺ and NO₃⁻ in plant roots under fluctuating proportions of both inorganic N forms.

On the other hand, fluxes of NH₄⁺ and NO₃⁻ 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 NO₃⁻ is transported across the plasma membrane via NO₃⁻/H⁺ symporters with the involvement of PM-H⁺-ATPase [13]. The concentration of NH₄⁺ can increase the activity of PM-H⁺-ATPase [14]. Furthermore, the expression of genes encoding PM-H⁺-ATPase was positively associated with fluxes of NH₄⁺ and NO₃⁻ [8]. The significant correlations between NH₄⁺, NO₃⁻ 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 NH₄⁺ and NO₃⁻ as well as their correlation with H⁺ flux in fine roots. In this study, a non-invasive micro-electrodes technique was employed to investigate NH₄⁺, NO₃⁻ 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 NH₄⁺ and NO₃⁻ fluxes; (ii) to illustrate the characteristics of NH₄⁺, NO₃⁻ and H⁺ fluxes and their interactions under different N forms and proportions.

2. Materials and Methods

2.1. 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° 03′ N, 118° 49′ E), under normal growth conditions (approximately 25 °C) in a photoperiod of 14/10 h of light/dark. Two months later, cuttings with uniform size and development were selected for the NMT experiments.

Seeds were collected from a healthy T. distichum grown in the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences. They were planted in black plastic pots (5 × 5 × 15 cm) filled with 1:1 volume of peat:perlite in a climate chamber (23–25 °C/15–18 °C, day/night; light per day, 14 h; photosynthetic photon flux, 160 μmol m⁻² s⁻¹; relative air humidity, 50–60%). After 19 weeks of growth, the plants were transferred into black plastic boxes (25 × 15 × 14.5 cm, 4 plants per box) containing 4 L of modified 1/4 Hoagland’s nutrient solution [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 ¹⁵N Atom% and enzymatic activities.

2.2. 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 investigate the net fluxes of NH₄⁺ and NO₃⁻ under different N forms and proportions, the measuring solutions were designed as (1) NH₄⁺ (NH₄Cl): 0.1 and 1.0 mM, (2) NO₃⁻ (KNO₃): 0.1 and 1.0 mM and (3) NH₄⁺:NO₃⁻: 1:3, 1:1, and 3:1 (total N = 2 mM), containing 0.5 mM MES, pH 6.0.

To explore the biomass, ¹⁵N 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 ¹⁵NH₄Cl and K¹⁵NO₃ (serving as control, CK), 1 mM ¹⁵NH₄Cl and 1 mM K¹⁵NO₃ 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 ¹⁵N Atom%, and the remaining 4 plants in each treatment were used for enzymatic activities.

2.3. 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 non-invasive 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 H⁺ electrode, 15 mM NaCl and 40 mM KH₂PO₄ 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 NH₄⁺ calibration, 0.05/0.5 mM NH₄Cl in addition to other compounds (0.5 mM MES, pH 6.4/5.4) were used in the measuring solution; for NO₃⁻ calibration, 0.05/0.5 mM KNO₃ 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 NH₄⁺/NO₃⁻ 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.

2.4. Determination of ¹⁵N Uptake and Enzyme Activities

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

Activities of nitrate reductase (NR, EC 1.7.99.4), nitrite reductase (NiR, EC 1.7.2.1), glutamine synthetase (GS, EC 6.3.1.2), glutamate synthetase (GOGAT, EC 1.4.7.1) and glutamate dehydrogenase (GDH, EC 1.4.1.2) in the roots were assayed [4].

2.5. Statistical Analysis

In order to determine the NH₄⁺, NO₃⁻ 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, ¹⁵N 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.

3. Results

3.1. Net Fluxes of NH₄⁺ and NO₃⁻ along the Root Tip

Net fluxes of NH₄⁺ and NO₃⁻ 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,b). The NH₄⁺ fluxes ranged from 17.02 (net influx) to 88.89 (net influx) pmol cm⁻² s⁻¹ in T. ascendens, and varied dramatically from −30.52 to 167.15 pmol cm⁻² s⁻¹ in T. distichum, and fluctuated between −90.94 and 46.31 pmol cm⁻² s⁻¹ in T. mucronatum when supplied as 1 mM NH₄Cl (Figure 2a). Intriguingly, both T. ascendens and T. distichum showed strong NH₄⁺ uptake rates from 2.1 to 3.0 mm along the root tip (Figure 2a).

Net NO₃⁻ fluxes ranged from −49.68 to 102.15 pmol cm⁻² s⁻¹ in T. ascendens, from −80.61 to 185.98 pmol cm⁻² s⁻¹ in T. distichum, and from −7.88 to 112.27 pmol cm⁻² s⁻¹ in T. mucronatum when fed with 1 mM KNO₃ (Figure 2b). The maximal net NO₃⁻ influxes of T. ascendens, T. distichum, and T. mucronatum were detected at 15.0, 3.0, and 5.0 mm from the root apex, respectively (Figure 2b). As a result, the following experiments selected 2.5 mm from the root apex as the specific position to investigate the net fluxes of NH₄⁺ and NO₃⁻. Moreover, 2.5 mm from the apex belongs to the elongation zone of the root tip in Taxodium plants (Figure 1b).

3.2. Net Fluxes of NH₄⁺ and NO₃⁻ under Different N Forms

As NH₄Cl and KNO₃ were added separately, the NH₄⁺ and NO₃⁻ fluxes fluctuated widely for all tested plants at 2.5 mm from the root apex during a 5-min period (Figure 3). Both NH₄⁺ and NO₃⁻ fluxes of T. distichum and T. mucronatum showed a tendency towards net influx. T. ascendens, however, tended to show net efflux of NH₄⁺ and net influx of NO₃⁻ (Figure 3a,d,g). When supplied with mixed N (NH₄NO₃), stable fluxes of NH₄⁺ and NO₃⁻ were observed, and distinctly, NH₄⁺ fluxes were much greater than NO₃⁻ fluxes in all Taxodium plants (Figure 3b,e,h). Compared to 1 mM NH₄Cl, average net fluxes of NH₄⁺ were stimulated by 688%, 171%, and 762% under 1 mM NH₄NO₃ in roots of T. ascendens, T. distichum, and T. mucronatum, respectively (Figure 3c,f,i). Thus, the increase of NH₄⁺ fluxes was as follows: T. mucronatum > T. ascendens > T. distichum (Figure 3c,f,i). The same order was observed for the decreases in net NO₃⁻ fluxes, which were decreased by 314%, 220%, and 81.66% under 1 mM NH₄NO₃ compared with that under 1 mM KNO₃, respectively (Figure 3c,f,i).

3.3. 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 and 2.84 times under 1.0 mM NH₄Cl treatment in T. distichum and T. mucronatum, respectively (Figure 4a). Apart from T. ascendens and T. distichum treated with 0.1 mM KNO₃, all the plants displayed a tendency for net NO₃⁻ influx when supplied with 0.1 or 1.0 mM KNO₃ (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).

3.4. Net NH₄⁺ and NO₃⁻ Fluxes under Different N Proportions

Under 2.0 mM TN (total nitrogen) consisting of various proportions of NH₄Cl and KNO₃ (1:3, 1:1, and 3:1), T. ascendens, T. distichum, and T. mucronatum showed great diversities in the fluxes of NH₄⁺, NO₃⁻ and TN (Figure 5). The net influx of NH₄⁺ ranged from 88.00 to 1480.80 pmol cm⁻² s⁻¹ across the three tested species (Figure 5a). It was 0.96 and 10.15 times greater under NH₄: NO₃ at 1:1 and 3:1 than at 1:3 in T. ascendens, respectively (Figure 5a). Similarly, the net NH₄⁺ influxes were 5.21 and 7.76 times higher than 1:3 when treated with 1:1 and 3:1, respectively, in T. mucronatum (Figure 5a). A decreasing trend of net NH₄⁺ influx was observed for T. distichum with an increase in the NH₄⁺ proportion (Figure 5a).

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).

3.5. ¹⁵N AT%, Ndff% and Enzyme Activities in the Roots of T. distichum

Compared with CK, ¹⁵N AT% was elevated by 70.27% or 29.73% in 1 mM ¹⁵NH₄⁺-treated or 1 mM ¹⁵NO₃⁻-treated T.distichum roots (

Table 1. Biomass, ¹⁵N AT% and Ndff% in the roots of T. distichum under three N treatments.

Treatments	Root Biomass/g	15N AT%	Ndff%
CK	0.30 ± 0.01 bc	0.37 ± 0.00 c	/
1 mM 15NH4+	0.34 ± 0.02 ab	0.63 ± 0.03 a	2.65 ± 0.31 a
1 mM 15NO3−	0.26 ± 0.01 c	0.48 ± 0.04 b	1.16 ± 0.38 b

Different letters behind the values in the same column indicate significant differences among the treatments according to Duncan’s Multiple Range Test at 5% level. ¹⁵N AT%: ¹⁵N Atom; Ndff%:¹⁵N from N source.

). 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 ¹⁵NH₄⁺ or ¹⁵NO₃⁻ during the 3 d experiment period (Table 1).

¹⁵NH₄⁺ or ¹⁵NO₃⁻ fertilization also has positive impacts on the activities of N assimilation enzymes (

Table 2. Activities of N assimilation enzymes in the roots of T. distichum under three N treatments.

Treatments	NR Activity μmol h−1 mg−1 Protein	NiR Activity μmol h−1 mg−1 Protein	GS Activity μmol h−1 mg−1 Protein	GDH Activity μmol h−1 mg−1 Protein	GOGAT Activity μmol h−1 mg−1 Protein
CK	0.58 ± 0.02 c	2.43 ± 0.04 c	0.33 ± 0.00 b	1.04 ± 0.06 b	0.78 ± 0.03 b
1 mM 15NH4+	0.87 ± 0.02 b	3.25 ± 0.08 a	0.46 ± 0.22 a	1.54 ± 0.08 a	1.24 ± 0.03 a
1 mM 15NO3−	1.92 ± 0.04 a	2.72 ± 0.01 b	0.34 ± 0.01 b	1.01 ± 0.04 b	0.74 ± 0.01 b

Different letters behind the values in the same column indicate significant differences between the treatments according to Duncan’s Multiple Range Test at 5% level. NR: nitrate reductase; NiR: nitrite reductase; GS: glutamine synthetase; GDH: glutamate dehydrogenase; GOGAT: glutamate synthetase.

). 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 ¹⁵NH₄⁺-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 ¹⁵NO₃⁻-fed T. distichum roots (Table 2).

4. Discussion

4.1. Spatial Variability of Net NH₄⁺ and NO₃⁻ 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 NH₄⁺ and NO₃⁻ in different root zones [26,27,28]. Spatial variability of net NH₄⁺ and/or NO₃⁻ flux has been observed in fine roots of various plant species [29]. For example, maximal net NH₄⁺ 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 NH₄⁺ and NO₃⁻ influxes along the root axis was also observed in our research. The largest net influxes of NH₄⁺ and NO₃⁻ 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 NH₄⁺ and NO₃⁻ 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 NH₄⁺ fluxes were shown in the elongation zones [28,32]. Moreover, Phyllostachys edulis showed relatively higher net influxes of NH₄⁺ and NO₃⁻ at 2–5 mm from the root apex [30]. The net NH₄⁺ or NO₃⁻ 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 NH₄⁺ and NO₃⁻ uptake rates among the three Taxodium species.

4.2. Net NH₄⁺ and NO₃⁻ Fluxes under Single N Treatments

Generally, environmental N levels have a significant impact on the NH₄⁺ and NO₃⁻ fluxes of fine roots [4]. For instance, gradual increases in the fluxes of NH₄⁺ and/or NO₃⁻ 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]. In our study, except for T. ascendens under NH₄Cl and T. mucronatum under KNO₃ solutions, most outcomes showed elevated NH₄⁺ or NO₃⁻ uptake rates resulting from increasing NH₄Cl or KNO₃ supply. Consistently, higher ¹⁵N AT% and Ndff% were induced by ¹⁵NH₄⁺ or ¹⁵NO₃⁻ treatment in the roots of T. distichum. Moreover, ¹⁵NH₄⁺ or ¹⁵NO₃⁻ fertilization also brought about higher activities of NR, NiR, GS, GDH and GOGAT in the roots of T. distichum. These results suggest that N fertilization could be applied to stimulate NH₄⁺ and NO₃⁻ absorption and assimilation capacities for Taxodium plants in practice.

Although an increasing N supply is likely to enhance N uptake in most cases, the provision of just NH₄⁺ could lead to soil acidification [34]. In most cases, to maintain ion homeostasis, roots release H⁺ while absorbing NH₄⁺, 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 NO₃⁻, OH⁻ could be released, contributing to the increase of pH [39]. Thus, a balanced supply of NH₄⁺ and NO₃⁻ is expected to improve the N uptake of plants and the soil environment.

4.3. Net NH₄⁺ and NO₃⁻ Fluxes under Mixed N Treatments

Many studies had demonstrated that the uptake of NH₄⁺ and NO₃⁻ was affected by each other when both N forms were provided [1,7]. In this study, the presence of NO₃⁻ stimulated the uptake of NH₄⁺, whereas the net fluxes of NO₃⁻ were inhibited by NH₄⁺ 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 NH₄⁺ and NO₃⁻ might interact with each other under coexistence N forms. These results might be related to cytosolic NH₄⁺/NO₃⁻ thresholds [30]. In detail, more NH₄⁺ may be required for plant development when NO₃⁻ was provided, while pre-existing NH₄⁺ may reduce the thresholds of NO₃⁻ in the plant [1]. Considering that a higher net NH₄⁺ influx than NO₃⁻ was observed, it can be concluded that Taxodium plants show a preference for NH₄⁺. It is noted that when NH₄Cl or KNO₃ was solely supplied, the fluxes of NH₄⁺ or NO₃⁻ in the three species were erratic. However, stable net NH₄⁺ and NO₃⁻ fluxes were observed when NH₄⁺ and NO₃⁻ 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 NH₄⁺ and NO₃⁻, 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 NH₄⁺ in fine roots of Taxodium species was expected to occur when NH₄⁺ and NO₃⁻ were supplied in different proportions [37]. In the case of tea, the maximum net NH₄⁺ influx was observed when NH₄⁺:NO₃⁻ was 1:1, and the highest net NO₃⁻ influx occurred when NH₄⁺:NO₃⁻ was 1.2:1 [1]. In blueberry (Vaccinium corymbosum L.), the mRNA levels of ammonium transporter 3 (VcAMT3) involved in NH₄⁺ uptake as well as nitrate transporter 1.5 (VcNRT1.5) and VcNRT2 involved in NO₃⁻ uptake was highest when the NH₄⁺:NO₃⁻ 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 NH₄⁺ and NO₃⁻ were supplied equivalently [1,24,41,42]. In this study, the best uptake rates of N were found when NH₄⁺:NO₃⁻ was 3:1, 1:3, and 3:1 for T. ascendens, T. distichum, and T. mucronatum, respectively, which could provide an applicable proportion of NH₄⁺ and NO₃⁻ when producing special N fertilizer for the productivity of Taxodium plants. Additionally, the optimal equilibrium between NH₄⁺ and NO₃⁻ supply largely differed between the three Taxodium species, implying that the induction of N transport systems require distinct NH₄⁺ and NO₃⁻ ratios among these plants.

In the present study, we found that with the change in the proportion of NH₄⁺: NO₃⁻ (total N concentration: 2 mM), the NH₄⁺ influxes were improved more than NO₃⁻. This observation indicates a preference for NH₄⁺ over NO₃⁻, which is in good agreement with our previous outcomes. In most plant species, NH₄⁺ is first absorbed into cells and then directly converted to amino acids, whereas cytosolic NO₃⁻ is assimilated at a higher energy cost. It is reduced to NO₂⁻ with the help of nitrate reductase (NR) and is further converted into NH₄⁺ in plastids by nitrite reductase (NiR), which requires more energy than NH₄⁺ for both transportation and further reduction [43]. On the other hand, the flux discrepancies between NH₄⁺ and NO₃⁻ might result from the lower activity of NO₃⁻ transport systems affected by NH₄⁺, which reduces the expression of the NO₃⁻-related genes [1]. In blueberry plants, the expression of AMTs and NRTs was largely affected by the different ratios of NH₄⁺: NO₃⁻ [40]. A previous study has indicated that different AMTs determined the uptake of NH₄⁺ 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 NH₄⁺ and NO₃⁻ when supplied at different proportions might be related to the distinct energetic and biochemical characteristics of uptake and assimilation pathway between NH₄⁺ and NO₃⁻ in plant roots [4,5,24].

4.4. Net NH₄⁺ and NO₃⁻ Fluxes Associated with H⁺

In this study, the alteration of H⁺ fluxes was tightly associated with the variation in NH₄⁺ or NO₃⁻. Previous studies revealed that H⁺ fluxes might be correlated with the transport of NH₄⁺ and NO₃⁻, since NH₄⁺ is transported into root cells through a symporter (co-transport with H⁺) and/or a uniporter, and NO₃⁻ 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 NH₄⁺ or NO₃⁻ 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 NH₄⁺ and NO₃⁻. 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 NH₄⁺/NO₃⁻ 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 NH₄⁺/NO₃⁻ in Taxodium roots.

5. Conclusions

In summary, spatial variability of NH₄⁺ and NO₃⁻ fluxes was observed along fine roots of Taxodium plants, and T. ascendens and T. distichum had higher fluxes of NH₄⁺ and NO₃⁻ at 2.1–3.0 mm from the root apex. In most cases, net fluxes of NH₄⁺ and NO₃⁻ increased with the elevated single N levels. NH₄⁺ and NO₃⁻ affected each other when they were both supplied, and Taxodium plants preferred NH₄⁺. Higher net N influxes were found when NH₄⁺ and NO₃⁻ were simultaneously supplied than sole N treatments, especially in T. ascendens and T. mucronatum at 3:1 of NH₄⁺:NO₃⁻. Additionally, H⁺ fluxes were tightly correlated with net NH₄⁺ and NO₃⁻ fluxes. These findings are valuable for understanding the characteristics of NH₄⁺ and NO₃⁻ 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.