Characteristics of NH₄⁺ and NO₃⁻ fluxes in tea (Camellia sinensis) roots measured by scanning ion-selective electrode technique

Abstract

As a vital beverage crop, tea has been extensively planted in tropical and subtropical regions. Nitrogen (N) levels and forms are closely related to tea quality. Based on different N levels and forms, we studied changes in NO₃⁻ and NH₄⁺ fluxes in tea roots utilizing scanning ion-selective electrode technique. Our results showed that under both single and mixed N forms, influx rates of NO₃⁻ were much lower than those of NH₄⁺, suggesting a preference for NH₄⁺ in tea. With the increase in N concentration, the influx rate of NO₃⁻ increased more than that of NH₄⁺. The NH₄⁺ influx rates in a solution without NO₃⁻ were much higher than those in a solution with NO₃⁻, while the NO₃⁻ influx rates in a solution without NH₄⁺ were much lower than those in a solution with NH₄⁺. We concluded that (1) tea roots showed a preference for NH₄⁺, (2) presence of NO₃⁻ had a negative effect on NH₄⁺ influx, and (3) NH₄⁺ had a positive effect on NO₃⁻ influx. Our findings not only may help advance hydroponic tea experiments but also may be used to develop efficient fertilization protocols for soil-grown tea in the future.

Introduction

As a crucial component of chlorophylls, nucleic acids, proteins and a great number of secondary plant metabolites, nitrogen (N) is essential for the growth of plants. Nitrate (NO₃⁻) and ammonium (NH₄⁺) are two major inorganic N forms for plants in soils. Due to various factors (e.g., root interference, soil moisture, soil microorganisms, etc.), the reciprocal transformation between ammonium and nitrate is very common in soils¹. Thus, roots are always ready to absorb both forms of nitrogen in soils. Both ions can be absorbed and used by plants because root cells possess transport systems such as nitrate and ammonium transporters². NO₃⁻ and NH₄⁺ have different biochemical and energetic features for assimilation, leading to various net fluxes of NO₃⁻/NH₄⁺ and ion preferences of plants, although both ions can be used by plants³.

Comparative studies on net fluxes of NH₄⁺ and NO₃⁻ have been conducted in different plants, and the preference for NH₄⁺ or NO₃⁻ is usually associated with the physiological needs of plants in various ecosystems⁴. Tea is an important beverage crop that has been extensively planted in tropical and subtropical regions. In tea plants, N levels and forms, especially in young shoots, are associated with the quality of tea. Previous research has demonstrated that tea plants have a higher absorption of NH₄⁺ compared to NO₃^− 5. However, high concentrations of NH₄⁺ are toxic in a majority of plants, including woody plants. If only NO₃⁻ or both ions are provided, no detrimental influences can be detected in plants^6,7. Little information can be found on the interactions between NH₄⁺ and NO₃⁻ fluxes in tea roots, although the uptake of NH₄⁺ and NO₃⁻ in tea has been explored extensively^4,5,8. Moreover, most previous studies on N uptake were carried out using an ¹⁵N labeling method, which was unable to interpret the dynamic processes of NH₄⁺ and NO₃⁻ fluxes^9,10.

Taking an electrophysiological approach, scanning ion-selective electrode technique (SIET) can evaluate ion/molecule-specific activities non-invasively¹¹. To date, NH₄⁺, NO₃⁻, H⁺, Cd²⁺, Ca²⁺, Mg²⁺, Na⁺, Cl⁻, K⁺, O₂ and Al³⁺ have been identified utilizing SIET; however, the application of SIET for the examinations of net NH₄⁺ and NO₃⁻ fluxes in tea roots has not yet been reported.

In this study, the fluxes of net NH₄⁺ and NO₃⁻ in absorbing tea roots exposed to various N forms were evaluated with SIET non-invasively. This research had the following objectivities: (1) to monitor any alterations in net NH₄⁺ and NO₃⁻ fluxes in tea roots under different N forms, and (2) to assess the interaction between fluxes of NH₄⁺ and NO₃⁻ in tea roots. This research is the first attempt to identify fluxes of net NH₄⁺ and NO₃⁻ in the presence of different N forms and interactions between NH₄⁺ and NO₃⁻ fluxes in tea roots utilizing SIET. Our findings may help advance hydroponic tea experiments and effective fertilization protocols for future soil-grown tea plants.

Results

Net fluxes of NO₃⁻ and NH₄⁺ under different N forms and levels

Tea roots were immersed in measuring solutions with different N forms (1 mM NH₄NO₃, 2 mM KNO₃ or 1 mM (NH₄)₂SO₄) to monitor the net fluxes of NO₃⁻ and NH₄⁺ under various N forms. Net flux curves of NH₄⁺ and NO₃⁻ are shown in Fig. 1. After the 7 d N starvation treatment, both NH₄⁺ and NO₃⁻ presented influx states on the root surface when different N forms were given. In addition, the influx rates of NO₃⁻ and NH₄⁺ improved gradually. In comparison to the treatments of a single N form, the influx rates of NH₄⁺ and NO₃⁻ under the NH₄NO₃ treatment were more stable (Fig. 1a,b). The influx rates of NO₃⁻ were lower than those of NH₄⁺ under both single and mixed N form treatments, which suggested that tea roots had a preference for NH₄⁺ (Fig. 1a,b). When N levels were the same, the total N influx rate of the NH₄NO₃ treatment was considerably higher than that of the single N form treatments (Fig. 1c).

Figure 1

Net fluxes of NO₃⁻ and NH₄⁺ on surfaces of tea roots under different N forms.

Net fluxes of NO₃⁻ and NH₄⁺ on tea root surfaces under single (a) and mixed N forms (b). Total N influx rates under the different N forms (c). The mean ± SE (n = 6) is shown in the data. In order to eliminate the “noise” caused by the oscillation, not only 6 biological repetitions, but also 70 measurement time points in each repetition were considered. Thus, SE = SD/√420. The different letters indicate differences between means at P < 0.05.

The influx rates of NO₃⁻ and NH₄⁺ under various proportions of N sources are shown in Fig. 2. With an increase in the NH₄⁺ concentration, the influx rates of NH₄⁺ first increased and then decreased under the same concentration of NO₃⁻ (Fig. 2). The highest influx rate of NH₄⁺ appeared when the ratio of NH₄⁺:NO₃⁻ was 1:1. For the influx rates of NO₃⁻, the highest influx rate of NO₃⁻ appeared when the ratio of NO₃⁻:NH₄⁺ was 1.2:1. This suggested that NH₄⁺:NO₃⁻ at 1:1 was the critical point and that the absorption rate of NH₄⁺ might not improve with an increase of NH₄⁺; meanwhile, the variation of the NO₃⁻ influx rate was extremely different.

Figure 2

Influx rates of NH₄⁺ and NO₃⁻ on tea root surfaces under different proportion of N sources.

The mean ± SE (n = 6) is shown in the data. The different letters indicate differences between means at P < 0.05.

The influx rates of NH₄⁺ and NO₃⁻ under different N levels are shown in Fig. 3. The influx rates of NH₄⁺ were 6.69 and 1.87 times higher compared to NO₃⁻ at 0.2 and 1.2 mM N levels, respectively. With increasing N concentration, the influx rates of NO₃⁻ and NH₄⁺ improved significantly. In addition, the influx rate of NO₃⁻ improved more than NH₄⁺ with the increase in N concentration. Although a high concentration of ammonium N is toxic for a majority of plants (including woody plants), the high concentration of NH₄⁺ (1.2 mM) did not affect tea tree growth in this study (Supplementary Figures S1 and 2). From phenotyping data, biomass and N contents of root, stem and leaf were higher with the supply of NH₄⁺-N compared to the supply of NO₃⁻-N. Thus, tea trees had better growth with the supply of NH₄⁺-N compared to the supply of NO₃⁻-N (Supplementary Figure S1). In addition, the content of total free amino acid in the supply of NH₄⁺-N was higher compared to the supply of NO₃⁻-N (Supplementary Figure S2). The main amino acids of the tea tree (such as aspartic acid, glutamic acid and theanine) were higher with the supply of NH₄⁺-N compared to the supply of NO₃⁻-N (Supplementary Figure S2). This suggested that tea roots had a stronger NH₄⁺ uptake ability, especially under low N conditions.

Figure 3

Influx rates of NH₄⁺ and NO₃⁻ on tea root surfaces under different N levels.

The mean ± SE (n = 6) is shown in the data. The different letters indicate differences between means at P < 0.05.

Interactions between NH₄⁺ and NO₃⁻ fluxes in tea roots

Changes in NH₄⁺ flux are shown in Fig. 4 after adding NH₄⁺ to the bathing solution either with or without NO₃⁻. NH₄⁺ presented influx states on the root surface regardless of whether the bathing solution had NO₃⁻. The NH₄⁺ influx rates in the bathing solution without NO₃⁻ were much higher compared with those in a solution with NO₃⁻. In plants without NO₃⁻ supply, the influx rates of NH₄⁺ increased and peaked approximately 200 s after NH₄⁺ addition (T1 stage), suggesting a vibrant status of the influx system without the NO₃^– supply and the ability to retain cytoplasmic NH₄⁺ to a specific degree in tea roots. The influx rates of NH₄⁺ remained stable (T2 stage), demonstrating that the NH₄⁺ influx and efflux systems had reached a balance and that NH₄⁺ influx was dominant. In plants with the NO₃⁻ supply, the influx rates of NH₄⁺ did not increase; however, they stabilized quickly and maintained the rate (t1 stage). Approximately 800 s after NH₄⁺ addition, the influx rates of NH₄⁺ began to decrease (t2 stage). The NH₄⁺ influx rates in the bathing solution with or without K⁺ are shown in Supplementary Figure S3. There was little difference between the NH₄⁺ influx rates in the bathing solution with or without K⁺, indicating that adding K⁺ had little effect on the net flux of NH₄⁺ in this study.

Figure 4

Influence of NO₃⁻ on NH₄⁺ net fluxes on tea root surfaces.

After adding (NH₄)₂SO₄ to the bathing solution without or with 1 mM KNO₃, the changes in tea root NH₄⁺ net fluxes (averaged over 49 s) are presented. The mean ± SE of NH₄⁺ influxes during the measurement period are shown (n = 6). (NH₄)₂SO₄ was added at the vertical arrows.

Changes in NO₃⁻ flux are shown in Fig. 5 after adding NO₃⁻ to the bathing solution without or with NH₄⁺. NO₃⁻ presented influx states on the root surface regardless of whether the bathing solution had NH₄⁺. The NO₃⁻ influx rates in the bathing solution without NH₄⁺ were much lower compared to those in a solution with NO₃⁻, which were just the opposite of the NH₄⁺ influxes above. In plants without the NH₄⁺ supply, the influx rates of NO₃⁻ remained stable. However, with the NH₄⁺ supply, the influx rates of NO₃⁻ could be divided into three stages: (1) a decrease with the lowest point approximately 400 s after NH₄⁺ addition (T1 stage); (2) a spiral increase from approximately 400 to 1200 s after NH₄⁺ addition (T2 stage); and (3) a slope decrease (T3 stage). The influx rates of NO₃⁻ were more unstable compared to NH₄⁺, especially in the bathing solution with NH₄⁺.

Figure 5

Influence of NH₄⁺ on NO₃⁻ net fluxes on tea root surfaces.

Variations of NO₃⁻ net fluxes in tea roots (averaged over 49 s) after adding KNO₃ to the bathing solution without or with 0.5 mM (NH₄)₂SO₄ are presented. The mean ± SE of NO₃⁻ influxes during the measurement period are shown (n = 6). KNO₃ was added at the vertical arrows.

Discussion

In all treatments, tea roots showed absorption states of NH₄⁺ and NO₃⁻, showing that the thresholds for plant development were higher than the cytosolic concentrations of NH₄⁺ and NO₃ after a 7 d N deprivation. Tea roots needed to maintain a certain level of NH₄⁺ or NO₃⁻ in the cytoplasm⁷. Additionally, tea roots showed a preference for NH₄⁺ when NH₄⁺ and NO₃⁻ existed at the same time. Greater net uptake of NH₄⁺ compared to net uptake of NO₃⁻ were reported in maize, rice and wheat roots when NH₄⁺ and NO₃⁻ were supplied simultaneously^12,13,14. In comparison to NO₃⁻ influx, there were some possible reasons for the observed preference for NH₄⁺ influx. As various root tissues needed various amounts of NH₄⁺ and NO₃⁻, one reason might involve root morphology. A higher concentration of NH₄⁺ was needed for protein synthesis in the meristem zone¹². In addition, NH₄⁺ absorbed by plants was transformed to amino acids directly in the roots, which required less energy and reducing equivalents for assimilation and transportation in most species^15,16. For tea plants, ammonia supplied to the tea roots was quickly stored as theanine, glutamine and arginine in the roots and leaves before the sprouting new shoots¹⁷. Previous research¹⁸ has reported that after tea plants were fed with ¹⁵N-NO₃⁻ and ¹⁵N-NH₄⁺, the amount of total amino acid in the xylem sap significantly increased, and those fed with ¹⁵N-NH₄⁺ had a greater increase compared to those fed ¹⁵N-NO₃⁻. Different from other plants, tea can turn redundant glutamic acid into theanine, which was a peculiar amino acid in tea¹⁹. Moreover, NH₄⁺ was more readily assimilated than NO₃⁻ into theanine²⁰. This process might have eliminated NH₄⁺ toxicity in tea roots and have created a NH₄⁺ preference in tea⁵.

Previous studies demonstrated that crop growth and yield were significantly improved when two forms of nitrogen were supplied at the same time. In this study, the highest total N influx rates were observed with the NH₄NO₃ treatment when N levels were the same, which suggested that tea roots had the highest nitrogen absorption efficiency when two forms of nitrogen were supplied simultaneously. This result was consistent with other plants reported^14,21,22. For most plants, roots released H⁺ after absorption of NH₄⁺, leading to a decreased pH in the growth medium, while roots released OH⁻ after absorption of NO₃⁻, leading to the increased pH in the growth medium^23,24,25. A mixed application of NO₃⁻ and NH₄⁺ at a 1:1 ratio encouraged higher foliar N content and glutamine synthetase (GS) and glutamate synthase (GOGAT) activity in tea²⁶. Although tea roots preferred NH₄⁺, a single application of ammonium nitrogen could aggravate soil acidification in tea gardens²⁷, and could significantly raise the amount of aluminum taken up by tea plants, leading to a decrease in tea quality²⁸. Therefore, to get a higher N absorption efficiency in tea and reduce soil acidification in tea gardens, two forms of nitrogen should be supplied simultaneously.

With the increase in N concentration, the influx rate of NO₃⁻ was improved more than NH₄⁺, which might be contributed to differences in activities and expressions of the transport systems between the two ions. Net NO₃⁻ and NH₄⁺ absorption can be regulated by low-affinity (LATS) and high-affinity transporters (HATS). When the exterior NH₄⁺ concentration was below 1 mM, HATS played a leading role in the uptake of NH₄⁺ absorption, and when the exterior NH₄⁺ concentration was above 1 mM, LATS were activated²⁹. While HATS played a main role in regulating NO₃⁻ uptake when the external NO₃⁻ concentration was below 1 mM, LATS started to work when the external NO₃⁻ concentration was above 0.5 mM³⁰. According to previous studies, with the increase in N concentration, the LATS for NO₃⁻ were stimulated much earlier than the LATS for NH₄⁺. In addition, Glass et al.³¹ and Britto et al.³² reported that transport through the low-affinity systems were poorly regulated when the high-affinity NH₄⁺ fluxes were effectively regulated. This might lead to the massive vain cycling of NH₄⁺ across the plasma membrane and toxic effects of superfluous NH₄⁺ accumulation. Thus, the influx rate of NO₃⁻ was improved more than NH₄⁺ with the increase in N concentration. The present data showed that the influx rate of NO₃⁻ was significantly lower than the influx rate of NH₄+ under low N conditions (0.2 mM N), which might be contributed to a lower energy cost for both transport and assimilation of NH₄^+ 16.

The present data demonstrates that the presence of NO₃⁻ had a negative effect on net NH₄⁺ uptake. Before being assimilated by plants, NO₃⁻ was restored as NH₄⁺, leading to the increase NH₄⁺ concentration in the cytoplasm. The HATS of NH₄⁺ were influenced by the negative feedback regulations and an increased cytosolic NH₄⁺ concentration suppressed the root influx of NH₄^+ 31. As widely acknowledged, NO₃⁻ is a mobile ion and can be restored both in the roots and leaf. Nitrate in tea roots can be directly transported to the xylem sap and then to the leaf¹⁸. When NO₃⁻ was restored as NH₄⁺ in the leaf, the concentration of NH₄⁺ in the leaf would increase. A shoot-to-root signal might be regarded as the effect of the local N status that controls the influx of NH₄^+ 33. In addition, NO₃⁻ had an inhibitory influence on GS enzyme activity, which might also a reason for the negative effect of NO₃⁻ on NH₄⁺ influx^34,35 (Fig. 6).

Figure 6

Proposed mechanisms of interaction between NH₄⁺ and NO₃⁻ fluxes in tea roots.

The absorption and transformation processes of NH₄⁺ and NO₃⁻ are shown. Some influence factors of NH₄⁺ and NO₃⁻ absorption are listed to explain possible mechanisms of interaction between the NH₄⁺ and NO₃⁻ fluxes in tea roots.

In contrast, the presence of NH₄⁺ had a positive effect on net NO₃⁻ uptake, which was consistent with previous studies performed in other species^14,33,36. There were several reasons to support this result. First, NH₄⁺ has been reported to increase the respiration rate of plants, which can provide energy for NO₃⁻ uptake³⁷. Second, the balance of H⁺, NH₄⁺ and NO₃⁻ could be used to explain this result. Tea roots took up a large amount of NH₄⁺ during growth and later released H⁺ to maintain the charge balance in the plant body³⁸. According to our current understanding of NO₃⁻ transportation, NO₃⁻ influx occurs with one H⁺ symport, and two possible H⁺ ions promote the inward transportation of one NO₃⁻ ion, while the efflux of H⁺ is meant to balance the influx of NH₄⁺. According to previous studies, due to NH₄⁺ stimulation of H⁺ efflux, a stimulation of NO₃⁻ absorption by NH₄⁺ might increase the availability of H⁺ for co-transport^39,40. Moreover, NH₄⁺ could significantly increase the activities of the GS and GOGAT enzymes, which plays an important role in nitrate reduction and nitrogen assimilation, providing material bases for NO₃⁻ absorption³⁴. In our study, the NO₃⁻ influx rates were more irregular in the various treatments. This was because NO₃⁻ could develop the functions of a mobile ion and an osmoticum¹⁴ (Fig. 6).

In conclusion, the elucidation of the mechanisms related to N transport is difficult when assessing net N flux. Net N flux is established as the total of N efflux and influx. Additionally, net N flux is affected by transportation and assimilation rates. The findings showed that tea roots presented influx states of NH₄⁺ and NO₃⁻ after a 7 d N-starvation. The uptake rates of NH₄⁺ in tea plants were higher than those of NO₃⁻. NH₄⁺-N can make tea trees grow better when only one single N source can be provided. Furthermore, the presence of NO₃⁻ had a negative effect on net NH₄⁺ influx, while NH₄⁺ had a positive influence on net NO₃⁻ influx. These findings may not only help guide further hydroponic experiments with tea but also help in developing efficient fertilization protocols for field-grown tea.

Methods

Plant materials and cultivation

Camellia sinensis var. Longjing 43 was used in this study. Annual cutting seedlings of Longjing 43 were transplanted to a full-strength nutrient solution for 75 d. The full-strength nutrient solution contained macronutrients (mmol L⁻¹) NH₄NO₃ (1), KH₂PO₄ (0.07), K₂SO₄ (0.3), MgSO₄·7H₂O (0.67), CaCl₂·2H₂O (0.53), and Al₂(SO₄)₃·18H₂O (0.035) and micronutrients (μmol L⁻¹) H₃BO₄ (7), MnSO₄·H₂O (1), ZnSO₄·7H₂O (0.67), CuSO₄·5H₂O (0.13), (NH₄)₆Mo₇O₂₄·4H₂O (0.047) and EDTA-Fe (4.2) at pH 5.0. The nutrient solution was circulated by pumps for 24 h every day and replaced every 3 days. Next, an N starvation treatment was carried out for 7 d. The N starvation treatment was conducted using the following nutrient solution which contained macronutrients (mmol L⁻¹) KH₂PO₄ (0.07), K₂SO₄ (0.3), MgSO₄·7H₂O (0.67), CaCl₂·2H₂O (0.53), and Al₂(SO₄)₃·18H₂O (0.035) and micronutrients (μmol L⁻¹) H₃BO₄ (7), MnSO₄·H₂O (1), ZnSO₄·7H₂O (0.67), CuSO₄·5H₂O (0.13), (NH₄)₆Mo₇O₂₄·4H₂O (0.047) and EDTA-Fe (4.2) at pH 5.0²⁰. The nutrient solution was circulated by pumps for 24 h every day and replaced every three days. After the 7 d N starvation treatment, the seedlings were harvested to measure ion fluxes.

Determinations of NO₃⁻ and NH₄⁺ fluxes at the root surface

The absorbing tea roots were chosen and cut off from the root system of every plant in every treatment group to evaluate the net fluxes of NO₃⁻ and NH₄⁺ in tea roots under various N forms. For the different nitrogen form treatments, tea roots were immersed in measuring solutions with different N forms (NH₄NO₃⁻N: 0.1 mM CaSO₄, 1 mM NH₄NO₃ and 0.3 mM MES; NH₄⁺-N: 0.1 mM CaSO₄, 1 mM (NH₄)₂SO₄, and 0.3 mM MES; and NO₃⁻-N: 0.1 mM CaSO₄, 2 mM KNO₃ and 0.3 mM MES). MES is 2-(N-morpholino)ethanesulfonic acid hydrate buffer. For different N level treatments, tea roots were soaked in measuring solutions with different N levels (0.2 mM NH₄⁺-N: 0.3 mM MES, 0.1 mM CaSO₄ and 0.1 mM (NH₄)₂SO₄; 1.2 mM NH₄⁺-N: 0.3 mM MES, 0.1 mM CaSO₄ and 0.6 mM (NH₄)₂SO₄; 0.2 mM NO₃⁻-N: 0.3 mM MES, 0.1 mM CaSO₄ and 0.2 mM KNO₃; and 1.2 mM NO₃⁻-N: 0.3 mM MES, 0.1 mM CaSO₄ and 1.2 mM KNO₃). Before analysis, tea roots were transferred to Petri dishes containing 10 mL of measuring solution and equilibrated for 10 min to reduce possible transition effects due to changes in the environmental conditions. Next, the equilibrated root was moved to another Petri dish containing fresh measuring solution and either NH₄⁺ or NO₃⁻ flux was measured utilizing the SIET technique. The coefficient of variation under different balance times were shown in Supplementary Figure S4. When the pretreatment time was 10 min, the coefficient of variations under the different treatments was lower and more stable. Therefore, a 10-min pretreatment time was enough and suitable for our study. Six repetitions were established for each treatment. In order to determine the area along the root axis corresponding with maximal net NH₄⁺ and NO₃⁻ influx, the net fluxes of both ions were measured along the root tips to an area located 40 mm from the apex (Figure S5). The maximum net NH₄⁺ and NO₃⁻ influxes occurred in area between 15 and 25 mm from the root apex. Thus, we chose area between 15 and 25 mm from the root apex as the measurement site. The measuring time of each root was 10 min. The SIET technique was used to measure the net ion flux (NMT-NRP-00A00 system, Younger USA Science and Technology Corporation). The SIET system and the corresponding application process have been previously described in detail for ion flux detection¹⁴. Briefly, ion-selective microelectrodes designed with 2–4 μm apertures were manufactured and silanized (for the NH₄⁺ electrode, 100 mM NH₄Cl was used as a backfilling solution, followed by a NH₄⁺ selective liquid ion exchange cocktail (#09879, Sigma); for the NO₃⁻ electrode, 10 mM KNO₃ was used as the backfilling solution, followed by a NO₃⁻ selective liquid ion exchange cocktail (#72549, Sigma)). Prior to performing the flux measurements, the microelectrodes were calibrated¹⁴.

The absorbing roots of tea were soaked in a test solution and excised from the root system to evaluate the effect of NO₃⁻ on NH₄⁺ flux (D (with NO₃⁻): 0.3 mM MES, 0.1 mM CaSO₄, 0.1 mM (NH₄)₂SO₄, and 1 mM KNO₃; E (without NO₃⁻): 0.3 mM MES, 0.1 mM CaSO₄ and 0.1 mM (NH₄)₂SO₄). NH₄⁺ flux was measured utilizing the SIET technique for 5 min after a 10-min balance in the measuring solution. Next, 0.5 mM (NH₄)₂SO₄ was added to the measuring solution. After each addition of (NH₄)₂SO₄, during the first 1–2 min, the measuring solution was mixed thoroughly by expelling and sucking it into a pipette 10 times. NH₄⁺ flux was measured using the SIET technique for another 25 min. The unstable data during the early stage were removed to gain ion flux curves.

The absorbing roots of tea were immersed in a measuring solution and excised from the root system to study the effect of NH₄⁺ on NO₃⁻ flux (F (with NH₄⁺): 0.3 mM MES, 0.1 mM CaSO₄, 0.2 mM KNO₃, and 0.5 mM (NH₄)₂SO₄; G (without NH₄⁺): 0.3 mM MES, 0.1 mM CaSO₄ and 0.2 mM KNO₃). NO₃⁻ flux was measured utilizing the SIET technique for 5 min after a 10-min balance in the measuring solution. Next, 1.0 mM KNO₃ was added to the measuring solution. The test process was the same as above.

Statistical analysis

To verify the importance of differences between treatments, one-way ANOVA was performed. Microsoft Excel (Microsoft Corporation, USA) and SPSS Window version 17 (SPSS Incorporation, Chicago, USA) were used to analyze data. To draw figures for the data, OriginPro 8.1 (Origin Incorporation, Chicago, USA) was utilized.