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Implication of quantifying nitrate utilization and CO2 assimilation of Brassica napus plantlets in vitro under variable ammonium/nitrate ratios

Abstract

Background

Plantlets grown in vitro with a mixed nitrogen source utilize sucrose and CO2 as carbon sources for growth. However, it is very difficult to obtain the correct utilization proportions of nitrate, ammonium, sucrose and CO2 for plantlets. Consequently, the biological effect of ammonium/nitrate utilization, the biological effect of sucrose/CO2 utilization, and the ammonium/nitrate use efficiency for new C input derived from CO2 assimilation/sucrose utilization are still unclear for plantlets.

Results

The bidirectional stable nitrogen isotope tracer technique quantified the proportions of assimilated nitrate and ammonium in Brassica napus plantlets grown at different ammonium/nitrate ratios. The utilization proportions of sucrose and CO2 could be quantified by a two end-member isotope mixing model for Bn plantlets grown at different ammonium/nitrate ratios. Under the condition that each treatment contained 20 mM ammonium, the proportion of assimilated nitrate did not show a linear increase with increasing nitrate concentration for Bn plantlets. Moreover, the proportion of assimilated CO2 did not show a linear relationship with the nitrate concentration for Bn plantlets. Increasing the nitrate concentration contributed to promoting the assimilation of ammonium and markedly enhanced the ammonium utilization coefficient for Bn plantlets. With increasing nitrate concentration, the amount of nitrogen in leaves derived from nitrate assimilation increased gradually, while the nitrate utilization coefficient underwent no distinct change for Bn plantlets.

Conclusions

Quantifying the utilization proportions of nitrate and ammonium can reveal the energy efficiency for N assimilation in plantlets grown in mixed N sources. Quantifying the utilization proportion of CO2 contributes to evaluating the photosynthetic capacity of plantlets grown with variable ammonium/nitrate ratios. Quantifying the utilization proportions of nitrate, ammonium, sucrose and CO2 can reveal the difference in the ammonium/nitrate use efficiency for new C input derived from CO2 assimilation/sucrose utilization for plantlets grown at variable ammonium/nitrate ratios.

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Background

Nitrate and ammonium are widely used in plant tissue culture. From the view of the energy cost of nitrogen (N) assimilation, ammonium might be regarded as the preferable N source because the nitrate reduction that reduces nitrate to ammonium consumes large amounts of reducing power [1]. However, excessive ammonium has detrimental effects on plant growth (ammonium toxicity) [1,2,3]. In general, ammonium toxicity can be alleviated by adding a small amount of nitrate [1, 2]. Hence, a combination of an appropriate nitrate concentration and a high concentration of ammonium will contribute to reducing the energy cost used for nitrogen assimilation.

Murashige and Skoog (MS) [4] medium, which has a high inorganic nitrogen concentration (60 mM), is widely used for most plant species. However, for some plant cultures, the amount of inorganic N in MS medium far exceeds the amount required for normal growth of plantlets in vitro [5, 6]. In addition, the ratio of ammonium to nitrate (1:2) in MS medium might not be optimal because Zhang et al. [6] found that the nitrogen in the leaves of plantlets was mainly derived from ammonium assimilation even if the concentration of nitrate was twice that of ammonium. Hence, excessive nitrate in MS medium is not optimal and causes a waste of inorganic N. Considering the fact that the concentration of nitrate was twice that of ammonium in MS medium (20 mM ammonium, 40 mM nitrate), optimizing the nitrate concentration will effectively improve the nitrogen use efficiency of plantlets. Moreover, when the ammonium concentration is fixed at 20 mM in culture medium, optimizing the nitrate concentration can provide a chance to understand the reason for the reported relief of nitrate-dependent ammonium toxicity.

During the multiplication stage, most plantlets utilize sucrose (usually 3% in MS medium, w/v) and CO2 as carbon (C) sources for mixotrophic growth, i.e., CO2 for autotrophic growth and sucrose for heterotrophic growth [7]. Hence, the new C input in plantlets is derived from CO2 assimilation and sucrose utilization. The proportion of assimilated CO2 can indicate the degree of photoautotrophy (i.e., the photosynthetic capacity) for plantlets [7]. Generally, the survival rate of plantlets during acclimation is positively correlated with their photosynthetic capacity [8]. Moreover, high photosynthetic capacity is usually accompanied by the production of more reducing power, which contributes to enhancing the assimilation of nitrate and ammonium. The growth status of plantlets is closely associated with their photosynthetic capacity. Hence, quantifying the proportion of assimilated CO2 will contribute to evaluating the photosynthetic capacity of plantlets.

The plant C/N ratio can be employed as a proxy measure of N-use efficiency (NUE) over time [9]. Generally, the C and N contents in leaves can be measured directly. Therefore, when the utilization proportions of nitrate, ammonium, sucrose and CO2 are known for plantlets, the C content derived from the CO2 assimilation/sucrose utilization can be obtained; the N content derived from the assimilation of nitrate/ammonium can also be obtained. As a result, the nitrate/ammonium use efficiency for the new C input derived from CO2 assimilation/sucrose utilization can be represented by the corresponding C/N ratio, which contributes to revealing the difference in the ammonium/nitrate use efficiency for new C input derived from CO2 assimilation/sucrose utilization for plantlets grown at variable ammonium/nitrate ratios. However, it is very difficult to quantify the utilization proportions of nitrate, ammonium, sucrose and CO2 by chemical methods.

The nitrogen isotope composition (δ15N) of plants is strongly connected to the δ15N of the culture substrate [10, 11]. Therefore, plant δ15N is widely used as an indicator of nitrogen sources [12,13,14]. Both nitrate reductase (NR) and glutamine synthetase (GS) discriminate against 15 N relative to 14 N [9, 15]. Hence, nitrogen isotope fractionation occurs during the assimilation of nitrate and ammonium. The nitrogen isotope discrimination of NR is in the range of 19–22‰ [15,16,17] or 26‰ [18], whereas the nitrogen isotope fractionation value of GS is 16.5 ± 1.5‰ [19]. The assimilation of inorganic nitrogen occurs in the roots and/or shoots depending on the plant species and available N form [20, 21]. As a result, the nitrogen isotope fractionation values of nitrate assimilation and ammonium assimilation are also difficult to obtain simultaneously. Hence, quantifying the proportion of assimilated nitrate and ammonium is not possible with the δ15N of plants when a single isotope tracer is used at near-natural abundance levels. However, the assimilation of nitrate and ammonium only occurred in leaves in this study because the concentrations of cytokinin and auxin in this experiment precluded root formation by the plantlets. As a result, the foliar δ15N values of the root-free plantlets were only derived from the mix of the δ15N values of assimilated nitrate and ammonium in leaves without interference from the assimilation of nitrate and ammonium in the roots. Moreover, the cloned plantlets had no individual differences and were maintained in the same culture conditions in this study. Hence, based on the bidirectional stable N isotope tracer technique [6], the proportion of nitrate and ammonium utilization can be quantified in root-free plantlets when two labeled stable nitrogen isotope treatments (the H and L treatments) are used. The only difference between the H and L treatments is in the δ15N value of the nitrate, a δ15N of 22.67‰ in H and of 8.08‰ in L.

The stable carbon isotope technique is commonly used to identify various carbon sources utilized by plants [7, 22, 23]. Generally, a two end-member isotope mixing model can be used to quantify the utilization proportion of two different carbon sources [23, 24]. In this study, the growth of plantlets depended on the CO2 assimilation and sucrose utilization. Therefore, the foliar carbon isotopic composition (δ13C) of plantlets is derived from the mix of the δ13C values of assimilated CO2 and utilized sucrose. Accordingly, based on a two end-member isotope mixing model, the foliar δ13C values of plantlets can be used to quantify the utilization proportion of sucrose/CO2 if the isotope fractionation values of sucrose and CO2 are obtained.

In the present study, plantlets of Brassica napus (Bn), which is characterized by a very high demand for N inputs in agricultural systems [25], were subjected to different inorganic N regimes where the concentration of ammonium was set as 20 mM in each treatment. The following were our main aims: (1) to reveal the difference in nitrate utilization in Bn plantlets grown in variable ammonium/nitrate ratios and (2) to quantify the ammonium/nitrate use efficiency for new C input derived from CO2 assimilation/sucrose utilization for plantlets grown at variable ammonium/nitrate ratios.

Methods

Plant materials and experimental treatments

Bn plantlets in vitro were employed as explants in this experiment. Single shoots of Bn plantlets were grown in culture media with four inorganic nitrogen regimes. The average fresh weight (FW) per shoot was 0.09 g for the Bn plantlets. Based on the ammonium concentration (20 mM) in the MS culture medium, the ammonium concentration was set as 20 mM in each treatment, and the nitrate concentrations in the four treatments were set at 5 mM, 10 mM, 20 mM and 40 mM. Accordingly, the ammonium:nitrate ratio was different in each treatment. Each inorganic nitrogen regime included two labeled stable nitrogen isotope treatments. The labeled treatments were separated into groups with high (H) and low (L) natural 15 N abundance in NaNO3, with a δ15N of 22.67‰ in H and of 8.08‰ in L. NH4Cl, with a δ15N of -2.64‰, was employed as the ammonium nitrogen in this experiment. Each Erlenmeyer flask (150 ml) contained 50 ml Murashige and Skoog (MS) [4] medium supplemented with 2.0 mg·L−1 6-benzylaminopurine, 0.2 mg·L−1 α-naphthylacetic acid, 3% (w/v) sucrose, and 7.5 g·L−1 agar. The Erlenmeyer flask was loosely closed with a piece of vented sealing film (vented membrane diameter available in 3 cm, pore size 0.2–0.3 μm), thus allowing gas exchange with the surrounding atmosphere. The concentrations of cytokinin and auxin in this experiment precluded root formation for Bn plantlets in vitro during the whole culturing stage. All culture media were adjusted to pH 5.8 and then autoclaved at 121 °C for 20 min. The Bn plantlets were maintained in a growth chamber with a 12-h photoperiod (50 μmol m−2 s−1 PPFD) at 25 ± 2 °C.

Determination of growth parameters

After 5 weeks of culturing, the Bn plantlets were removed from the Erlenmeyer flasks in the afternoon. The biomass of each Bn plantlet (FW) was measured. Additionally, the leaf biomass of each Bn plantlet was also measured. Next, the shoots of each Bn plantlet were counted. The leaves of the Bn plantlets were dried at 60 °C. The increase in biomass of each plantlet (Increased biomass) was calculated as the difference between the initial FW of the shoot and the plantlet biomass after culture for 5 weeks. Moreover, the leaf dry weight (DW) of each Bn plantlet was also measured [see Additional file 1]. Finally, the dried leaves were ground to a fine powder.

Chlorophyll concentration determination

A total of 0.1 g of fresh leaf that had been triturated in a mortar with a small amount of liquid nitrogen was macerated with 10 ml 95% ethanol for 24 h at 4 °C. The chlorophyll concentration in the extract was spectrophotometrically determined at 665 and 649 nm. The concentrations, including chlorophyll a and chlorophyll b concentrations, were determined on a fresh weight basis (mg·g–1) and calculated according to Alsaadawi et al. [26].

Analysis of elements and determination of δ15N and δ13C in plantlets

The total nitrogen and carbon contents of the dried leaves were determined using an elemental analyzer (vario MACRO cube, Germany). Both δ15N and δ13C were measured by a gas isotope ratio mass spectrometer (MAT-253, Germany). Isotope ratios were calculated as follows:

$$\updelta {\left[{}^{13}\mathrm{C},{}^{15}\mathrm{N}\right]}_{\mathrm{samples}}=\left({R}_{\mathrm{sample}}/{R}_{\mathrm{standard}}-1\right)\times 1000$$
(1)

where Rsample refers to the 13C/12C or 15N/14 N of the plant material, and Rstandard refers to the isotope ratio of a known standard (PDB or N2 in air). International isotope secondary standards of known 13C/12C ratios (IAEA CH3 and IAEA CH6) were used for calibration to a precision of 0.1‰. For nitrogen, isotope secondary standards of known 15N/14 N ratios (IAEA N1, IAEA N2, and IAEA NO3) were used to calibrate the instrument to reach a precision of 0.2‰ [27].

Quantification of the contributions of nitrate and ammonium to total inorganic nitrogen assimilation

The proportions of nitrate and ammonium assimilated by Bn plantlets were determined by the bidirectional stable nitrogen isotope tracer technique [6]. Thus, the proportion of assimilated nitrate (fA) contributing to total inorganic nitrogen assimilation could be calculated by the following equation:

$${f}_{\mathrm{A}}=\left({\updelta }_{\mathrm{T}H}-{\updelta }_{\mathrm{T}L}\right)/\left({\updelta }_{\mathrm{A}H}-{\updelta }_{\mathrm{A}L}\right)$$
(2)

where δTH is the foliar δ15N value of the plantlets cultured with mixed-nitrogen sources, whose δ15N of nitrate in culture media was 22.67‰. δTL is the foliar δ15N value of the plantlets cultured with mixed-nitrogen sources, whose δ15N of nitrate in culture media was 8.08‰. Accordingly, δAH and δAL are the δ15N values derived from nitrate assimilation. The proportion of assimilated ammonium (fB) contributing to total inorganic nitrogen assimilation was calculated using the following equation:

$${f}_{\mathrm{B}}=1-{f}_{\mathrm{A}}$$
(3)

The standard error (SE) of fA and fB was achieved by the error propagation formula.

In this study, δTH and δTL could be obtained directly. However, when the plantlets were cultured in the medium with mixed-nitrogen sources, it would have been difficult to directly obtain δAH and δAL, which are involved in nitrogen isotope discrimination in nitrate assimilation and the exchange of unassimilated nitrate between the shoot and the substrate during the whole culture period. Hence, δAL and δAH changed over time in this experiment. However, we were able to obtain δAL and δAH when the plantlets were grown in culture medium in which nitrate was the sole nitrogen source.

The δAL and δAH in nitrate-grown plantlets could be affected by unassimilated nitrate. However, a previous study found that the storage pool of nitrate in leaves of tomato and tobacco plants was replenished in the dark and became depleted in the light, and the foliar nitrate concentration of tomato and tobacco plants reached a low level in the afternoon [28, 29]. Hence, when the plantlets had been cultured for 5 weeks and harvested in the afternoon, the amount of unassimilated nitrate in the leaves of plantlets would be very small in comparison with the amount of assimilated nitrate. Moreover, the foliar δ15N value of Bn plantlets did not vary significantly among nitrate concentrations ranging from 10 to 40 mM [6, 30], which suggested that the effect of unassimilated nitrate in leaves on the foliar δ15N value could be neglected. As a result, the δAL and δAH of Bn plantlets grown in mixed-nitrogen sources could be replaced by the δAL and δAH in nitrate-grown Bn plantlets in this study.

Sodium nitrate with a δ15N of 22.67‰/8.08‰ was used as the sole nitrogen source in their study [6, 30]. Hence, the average foliar δ15N value in nitrate-grown Bn plantlets at the three nitrate supply levels (10, 20, and 40 mM) was approximately equal to the δ15N value (δAL or δAH) of Bn plantlets cultured in the medium with mixed-nitrogen sources in this study. As a result, we were able to obtain δAL and δAH. δAL was 3.17 ± 0.12‰ (n = 9, SE) for the L Bn plantlets [30], and δAH was 15.19 ± 0.29‰ (n = 9, SE) for the H Bn plantlets [6]. After determining δTH, δTL, δAH and δAL, we were able to calculate fA and fB. However, because the efflux of nitrate to the external media occurred during the whole culture period, we must acknowledge that end members δAL and δAH may change slightly if the proportional efflux of nitrate back to the media changes. In addition, based on the fact that the foliar δ15N value of Bn plantlets did not vary significantly among nitrate concentrations ranging from 10 to 40 mM [6, 30], the presence of ammonium was assumed to have no effect on net discrimination against nitrate in this study. Hence, the proportion of assimilated nitrate obtained by Eq. (2) might not be precise enough.

Quantifying the contribution of nitrate/ammonium utilization to the amount of nitrogen in leaves

The nitrogen accumulation amount (NAA) of the leaves was the absolute nitrogen content in the dried leaves and was calculated using the following equation:

$$\mathrm{NAA}=\left(\mathrm{DW}\times \mathrm{Ncontent}\right)/\mathrm{M}$$
(4)

where M is the molar mass of nitrogen, and the N content of the dried leaves was determined by an elemental analyzer.

The nitrogen in leaves was derived from the assimilation of nitrate and ammonium. Therefore, the amount of nitrogen in leaves derived from assimilated nitrate/ammonium could be calculated by the following equations:

$${\mathrm{NAA}}_{\mathrm{nitrate}}=\mathrm{NAA}\times {f}_{\mathrm{A}}$$
(5)
$${\mathrm{NAA}}_{\mathrm{ammonium}}=\mathrm{NAA}\times {f}_{\mathrm{B}}$$
(6)

where NAAnitrate is the amount of nitrogen in leaves derived from nitrate assimilation, and NAAammonium is the amount of nitrogen in leaves derived from ammonium assimilation. The standard error (SE) of NAAnitrate and NAAammonium was calculated by the error propagation formula.

Nitrogen utilization coefficient (NUC) of ammonium and nitrate

The nitrogen utilization coefficient (NUC) is the ratio of the total nitrogen content in the dried leaves relative to the nitrogen content in the medium. Therefore, the nitrogen utilization coefficient of ammonium (NUCammonium) and nitrate (NUCnitrate) could be calculated by the following equation:

$${\mathrm{NUC}}_{\mathrm{ammonium}}\left(\mathrm{\%}\right)=\left({\mathrm{NAA}}_{\mathrm{ammonium}}/{\mathrm{n}}_{\mathrm{ammonium}}\right)\times 100$$
(7)
$${\mathrm{NUC}}_{\mathrm{nitrate}}\left(\mathrm{\%}\right)=\left({\mathrm{NAA}}_{\mathrm{nitrate}}/{\mathrm{n}}_{\mathrm{nitrate}}\right)\times 100$$
(8)

where nammonium and nnitrate are the number of moles of ammonium and nitrate in the medium, respectively. The standard error (SE) of NUCammonium and NUCnitrate was calculated by the error propagation formula.

Quantifying the proportion of inorganic carbon utilization in Bn plantlets

In this study, the external C source apart from CO2 was the sucrose for Bn plantlets. Therefore, the foliar δ13C value of the Bn plantlet was derived from the mix of the δ13C values of assimilated inorganic and organic carbon. Based on a two end-member isotope mixing model [23, 24], an equation representing this utilization of two different carbon sources by Bn plantlets can be established as follows:

$${\updelta }_{\mathrm{T}}={f}_{\mathrm{P}}\times {\updelta }_{\mathrm{C}}+\left(1-{f}_{\mathrm{P}}\right)\times {\updelta }_{\mathrm{S}}$$
(9)

where δT is the foliar δ13C value of Bn plantlets grown in mixed-carbon sources and could be obtained directly. fP is the proportion of assimilated CO2. 1-fP is the proportion of utilized sucrose. δC is the δ13C value derived from CO2 assimilation. δS is the δ13C value derived from sucrose assimilation. Accordingly, Eq. (9) can be rewritten as Eq. (10):

$${f}_{\mathrm{P}}=\left({\updelta }_{\mathrm{T}}-{\updelta }_{\mathrm{S}}\right)/\left({\updelta }_{\mathrm{C}}-{\updelta }_{\mathrm{S}}\right)$$
(10)

The standard error (SE) of fP was achieved by the error propagation formula.

When the plantlets were grown in mixed-carbon sources, it would have been difficult to directly obtain δS and δC. To obtain δS, the Bn plantlets were cultured in a CO2-free atmosphere where CO2 was absorbed by soda lime, and sucrose was the only carbon source for Bn plantlets. As a result, the isotope fractionation value of sucrose assimilation could be obtained indirectly. The isotope fractionation value of sucrose assimilation was 2.54 ± 0.13‰ (n = 3, SE) for Bn plantlets.

Bn plantlets cannot survive without a supply of sucrose. Hence, the isotopic fractionation value of CO2 assimilation cannot be obtained directly for Bn plantlets. To obtain δC, the seeds of Bn were grown in MS culture medium (20 mM ammonium, 40 mM nitrate) without sucrose. The culture conditions for Bn seeds were exactly the same as those for the L and H treatments, i.e., they were grown at the same time in the same chamber in the same Erlenmeyer flasks, which were closed with the same vented sealing film. After 5 weeks of culturing, the leaves of Bn seedlings were harvested for the measurement of δ13C. The carbon in leaves was only derived from CO2 assimilation for Bn seedlings. Hence, the foliar δ13C value of the Bn seedling, derived from seed germination, could be used to approximate δC in this study. As a result, δC could be obtained indirectly and was -28.05 ± 0.13‰ (n = 3, SE) for Bn plantlets in this study. After δT, δS and δC were known, we were able to calculate fP. However, the supply of inorganic N might affect the photosynthetic capacity of plants. Plants with low photosynthetic capacity might be more depleted in 13C than plants with high photosynthetic capacity. Hence, we must acknowledge that the δC in Bn plantlets grown at a concentration below 60 mM inorganic N might be less than -28.05‰ in this study. Hence, the fP might be somewhat overestimated for Bn plantlets grown at a concentration below 60 mM inorganic N.

The C/N ratios of leaves

After determining the carbon (CT) and nitrogen (NT) contents of leaves, the CT/NT ratio of leaves could be obtained directly. In this study, we quantified the proportions of assimilated C and N in Bn plantlets. Accordingly, the carbon content derived from the CO2 assimilation (CC) and sucrose utilization (CS) could be obtained; the nitrogen content derived from the assimilation of nitrate (NN) and ammonium (NA) could also be obtained. As a result, the nitrate use efficiency for new C input derived from the CO2 assimilation (CC/NN ratio), the nitrate use efficiency for new C input derived from sucrose utilization (CS/NN ratio), the ammonium use efficiency for new C input derived from the CO2 assimilation (CC/NA ratio), and the ammonium use efficiency for new C input derived from sucrose utilization (CS/NA ratio) can be calculated by the following equations:

$${\mathrm{C}}_{\mathrm{C}}/{\mathrm{N}}_{\mathrm{N}} \mathrm{ratio }= \frac{{f}_{\mathrm{P} \times {\mathrm{C}}_{\mathrm{T}}}}{{f}_{\mathrm{A}} \times {\mathrm{N}}_{\mathrm{T}}} = \frac{{f}_{\mathrm{P}}}{{f}_{\mathrm{A}}} \times \frac{{\mathrm{C}}_{\mathrm{T}}}{{\mathrm{N}}_{\mathrm{T}}}$$
(11)
$${\mathrm{C}}_{\mathrm{S}}/{\mathrm{N}}_{\mathrm{N}} \mathrm{ratio} = \frac{\left(1-{f}_{\mathrm{P}}\right) \times {\mathrm{C}}_{\mathrm{T}}}{{f}_{\mathrm{A}} \times {\mathrm{N}}_{\mathrm{T}}} = \frac{1 - {f}_{\mathrm{P}}}{{f}_{\mathrm{A}}} \times \frac{{\mathrm{C}}_{\mathrm{T}}}{{\mathrm{N}}_{\mathrm{T}}}$$
(12)
$${\mathrm{C}}_{\mathrm{C}} /{\mathrm{N}}_{\mathrm{A}} \mathrm{ratio }= \frac{{f}_{\mathrm{P }\times {\mathrm{C}}_{\mathrm{T}}}}{\left(1 - {f}_{\mathrm{A}}\right) \times {\mathrm{N}}_{\mathrm{T}}} = \frac{{f}_{\mathrm{P}}}{1 - {f}_{\mathrm{A}}}\times \frac{{\mathrm{C}}_{\mathrm{T}}}{{\mathrm{N}}_{\mathrm{T}}}$$
(13)
$${\mathrm{C}}_{\mathrm{S}}/{\mathrm{N}}_{\mathrm{A}}\mathrm{ ratio}=\frac{\left(1-{f}_{\mathrm{P}}\right) \times {\mathrm{C}}_{\mathrm{T}}}{\left(1-{f}_{\mathrm{A}}\right) \times {\mathrm{N}}_{\mathrm{T}}}= \frac{1 - {f}_{\mathrm{P}}}{1 - {f}_{\mathrm{A}}} \times \frac{{\mathrm{C}}_{\mathrm{T}}}{{\mathrm{N}}_{\mathrm{T}}}$$
(14)

The standard error (SE) of the CC/NN ratio, CC/NA ratio, CS/NN ratio, and CS/NA ratio was achieved by the error propagation formula.

Statistical analysis

The data were subjected to analysis of variance (ANOVA). The means of the different groups were compared via Tukey’s test (p < 0.05). The data are shown as the mean ± standard error (SE).

Results

Growth

The nitrate concentration had a significant effect on the growth of Bn plantlets. As shown in Table 1, when the ammonium concentration remained at 20 mM in each treatment, increasing the supply of nitrate could promote the growth of Bn plantlets. In addition, the leaf biomass of Bn plantlets increased significantly with increasing nitrate supply. With respect to the proliferation of shoots, the Bn plantlets showed no significant difference with increasing nitrate concentration, except at the lowest concentrations. Generally, Bn plantlets had good performance with respect to shoot proliferation under all treatments (Table 1).

Table 1 The growth parameters of Brassica napus plantlets cultured under nitrate treatment

Chlorophyll concentrations

The chlorophyll concentration of the Bn plantlets was significantly affected by the nitrate supply. Under the condition that each treatment included 20 mM ammonium, the chlorophyll concentration of the Bn plantlets showed a positive response to increasing nitrate concentrations. Increasing the supply of nitrate could promote the biosynthesis of chlorophyll in Bn plantlets (Table 2).

Table 2 The chlorophyll concentration of Brassica napus plantlets cultured under nitrate treatment

Elemental analysis of the Bn plantlets

The foliar nitrogen content of Bn plantlets was above 5% in all treatments. Supplying a certain concentration of nitrate could not effectively increase the foliar nitrogen content for Bn plantlets. As shown in Fig. 1, the foliar nitrogen content of Bn plantlets was not significantly different when the nitrate supply ranged from 5 to 20 mM. Moreover, the leaf carbon content of Bn plantlets did not show a significant difference with increasing nitrate (Fig. 1).

Fig. 1
figure 1

Nitrogen content (a) and carbon content (b) of the Brassica napus plantlets cultured under nitrate treatment. Each nitrate treatment contained 20 mM ammonium. The nitrogen and carbon content was expressed as a percent of foliar dry weight, respectively. The mean ± SE (n = 3) followed by different letters in the same legend differ significantly (Tukey’s test, p < 0.05)

Foliar carbon isotope ratio of the Bn plantlets

The δ13C values of Bn plantlets only showed significant differences at the lowest nitrate concentration. As shown in Fig. 2, increasing the nitrate supply did not significantly affect the δ13C values of Bn plantlets when the nitrate concentration was in the range of 10 to 40 mM.

Fig. 2
figure 2

The δ13C values of Brassica napus plantlets in vitro cultured under nitrate treatment. Note: Each nitrate treatment contained 20 mM ammonium. The mean ± SE (n = 3) followed by different letters in the bar graph differ significantly (Tukey’s test, p < 0.05)

The proportion of CO2 and sucrose utilization by the Bn plantlets

Increasing the supply of nitrate contributed to enhancing the proportion of CO2 utilization for Bn plantlets (Fig. 3). However, when the supply of nitrate reached 10 mM, there was no higher assimilation of CO2 for Bn plantlets. The proportion of CO2 utilization was lower than that of sucrose utilization at all nitrate concentrations, which suggested that the sucrose utilization was predominant for Bn plantlets. Nonetheless, increasing the nitrate concentration could reduce the predominance of the sucrose utilization.

Fig. 3
figure 3

The proportion of CO2 (a) and sucrose (b) utilization by the Brassica napus plantlets cultured under nitrate treatment. Note: Each nitrate treatment contained 20 mM ammonium. The error bars were calculated by the error propagation formula

Foliar nitrogen isotope ratio of the Bn plantlets

The δ15N values of Bn plantlets cultured in the H and L treatments were very different at different nitrate concentrations (Fig. 4). The minimum δ15N values in the L treatment were approximately -3.0‰, which suggested that δ15N values of the ammonium assimilation tended to impoverish in Bn plantlets. The δ15N value of Bn plantlets was significantly affected by nitrate concentration in both the H and L treatments. Increasing the supply of nitrate contributed to enriching 15N in Bn plantlets.

Fig. 4
figure 4

The foliar δ15N values of the Brassica napus plantlets cultured under nitrate treatment. Note: (a) The foliar δ15N values of the Brassica napus plantlets in the L-labeled treatment. (b) The foliar δ.15 N values of the Brassica napus plantlets in the H-labeled treatment. Each nitrate treatment contained 20 mM ammonium. The mean ± SE (n = 3) followed by different letters in the same legend differ significantly (Tukey’s test, p < 0.05)

The contribution of nitrate/ammonium to total inorganic nitrogen assimilation

The proportion of assimilated nitrate did not show a linear increase with increasing nitrate concentration for Bn plantlets (Fig. 5). The proportion of assimilated ammonium showed an obvious downward trend for Bn plantlets when the nitrate concentration increased from 10 to 40 mM. The contribution of nitrate utilization to total inorganic nitrogen assimilation was distinctly lower than that of ammonium in all treatments. Ammonium assimilation was predominant for Bn plantlets grown in mixed N source.

Fig. 5
figure 5

The contribution of nitrate (a) and ammonium utilization (b) to total inorganic nitrogen assimilation in the Brassica napus plantlets cultured under nitrate treatment. Note: Each nitrate treatment contained 20 mM ammonium. The error bars were calculated by the error propagation formula

The contribution of nitrate/ammonium utilization to the amount of nitrogen in leaves

The amount of nitrogen in leaves (NAA) of Bn plantlets showed a positive response to increasing nitrate concentrations when each treatment contained 20 mM ammonium. As shown in Fig. 6, with increasing nitrate concentration, the amount of nitrogen in leaves derived from nitrate assimilation (NAAnitrate) gradually increased, and the amount of nitrogen in leaves derived from ammonium assimilation (NAAammonium) also increased. Increasing the supply of nitrate could simultaneously promote the assimilation of nitrate and ammonium. NAAammonium increased more than double when the nitrate concentration reached 10 mM.

Fig. 6
figure 6

The amount of nitrogen in leaves (NAA), the amount of nitrogen in leaves derived from nitrate assimilation (NAAnitrate), and the amount of nitrogen in leaves derived from ammonium assimilation (NAAammonium) in the Brassica napus plantlets cultured under nitrate treatment. Note: Each nitrate treatment contained 20 mM ammonium. The error bars of NAAnitrate and NAAammonium were calculated by the error propagation formula

The utilization coefficients of nitrate and ammonium of the Bn plantlets

The utilization coefficients of nitrate and ammonium of the Bn plantlets showed different responses to increasing nitrate concentrations when each treatment contained 20 mM ammonium. The nitrate utilization coefficients (NUCnitrate) of the Bn plantlets showed no distinct change with increasing nitrate concentration, while the ammonium utilization coefficients (NUCammonium) of the Bn plantlets increased obviously with increasing nitrate concentration (Fig. 7). Increasing the supply of nitrate could markedly enhance the NUCammonium of the Bn plantlets, which contributed to reducing futile ammonium cycling.

Fig. 7
figure 7

The utilization coefficients of nitrate (a) and ammonium (b) in the Brassica napus plantlets cultured under nitrate treatment. Note: Each nitrate treatment contained 20 mM ammonium. The error bars were calculated by the error propagation formula

The C/N ratios of leaves

There were clear differences between the CT/NT ratio, CC/NN ratio, CC/NA ratio, CS/NN ratio, and CS/NA ratio of leaves in Bn plantlets (Table 3). The CT/NT ratio of leaves tended to decrease with 40 mM nitrate. The CS/NN ratio and CS/NA ratio of leaves showed a downward trend with increasing nitrate concentration.

Table 3 The C/N ratios of leaves in Brassica napus plantlets cultured under nitrate treatment

With increasing nitrate concentration, the CC/NN ratio of leaves first increased and then decreased, while the CC/NA ratio of leaves slowly increased. As shown in Table 3, the CS/NN ratio of leaves was higher than the CS/NA ratio of leaves under each treatment; the CC/NN ratio of leaves was also higher than the CC/NA ratio of leaves under each treatment. Therefore, to some extent, the nitrate use efficiency for new C input was higher than that of ammonium.

Discussion

Plant δ15N is a physiological indicator of N demand and fractionation that reflects changes in metabolic N fluxes and/or environmental effects [31, 32]. Since the use of nitrate and ammonium by plants are different, they exhibit different values of δ15N depending on the N source [33, 34]. In this study, the δ15N values of the Bn plantlets showed large differences between the L- and H-labeled treatments (Fig. 4). The foliar δ15N value of Bn plantlets is derived from the mix of the δ15N values of assimilated nitrate and ammonium in the leaves because no root formation occurs in this experiment. In addition, the δ15N values of Bn plantlets in each treatment are different from those of the substrate, which suggests that nitrogen isotope fractionation occurs during the assimilation of inorganic nitrogen in the Bn plantlets [30]. Generally, both the efflux of nitrate and ammonium to the external media and the assimilation of nitrate and ammonium can affect nitrogen isotope discrimination [35]. Therefore, if we are able to obtain the nitrogen isotope fractionation values of assimilated nitrate and ammonium, it will be possible to quantify the proportion of assimilated nitrate/ammonium with the δ15N values of the root-free plantlets in the L- or H-labeled treatments. However, it is very difficult to simultaneously obtain the nitrogen isotope fractionation values of nitrate assimilation and ammonium assimilation when the plantlets are grown in a mixed-nitrogen source.

According to the bidirectional stable nitrogen isotope tracer technique [6], when two labeled stable nitrogen isotope treatments are used, it is unnecessary to simultaneously obtain the nitrogen isotope fractionation values of nitrate assimilation and ammonium assimilation. As shown in Eq. (2), the proportion of assimilated nitrate depends only on δTH, δTL, δAL and δAH. δTH and δTL are the foliar δ15N values of the Bn plantlets grown in the mixed-nitrogen source and can be obtained directly. δAL and δAH can be replaced by the foliar δ15N values of the plantlets grown in the corresponding culture medium in which nitrate is the sole nitrogen source. Hence, when δTH, δTL, δAH and δAL are determined, the proportion of assimilated nitrate/ammonium can be quantified for Bn plantlets. Meanwhile, the proportion of assimilated sucrose and CO2 can be quantified by Eq. (10) for Bn plantlets. As a result, the utilization proportions of nitrate, ammonium, CO2 and sucrose can be obtained simultaneously for Bn plantlets. The total nitrogen and carbon contents of leaves of Bn plantlets can be determined by an elemental analyzer in this study. Hence, the nitrate/ammonium use efficiency for new C input derived from CO2 assimilation/sucrose utilization can be represented by the corresponding C/N ratio for Bn plantlets [9].

Generally, an excessive nitrogen supply is usually accompanied by low nitrogen use efficiency [36, 37]. As shown in Table 3, the nitrate use efficiency for new C input derived from the CO2 assimilation (as indicated by the CC/NN ratio) was the lowest for Bn plantlets when the nitrate concentration increased to 40 mM. Moreover, the nitrate use efficiency for new C input derived from sucrose utilization (as indicated by the CS/NN ratio) was also the lowest for Bn plantlets when the nitrate concentration increased to 40 mM. These results indicate that excessive nitrate supply is not optimal for Bn plantlets. The excess of nitrate affects the assimilation of C, which results in a decrease in the nitrate use efficiency for new C input. Interestingly, the maximum nitrate use efficiency for new C input derived from the CO2 assimilation was not achieved at the lowest nitrate concentration for Bn plantlets. The CC/NN ratio of Bn plantlets depended on the fP, fA, and CT/NT ratios (Eq. 11) in this study. There was little difference in the CT/NT ratio of Bn plantlets when the nitrate concentration was in the range of 5 to 20 mM. Hence, the CC/NN ratio of Bn plantlets was mainly dependent on fP and fA when the nitrate concentration was in the range of 5 to 20 mM. As shown in Fig. 3, the fP of Bn plantlets showed an obvious increase when the nitrate concentration increased from 5 to 10 mM. At the same time, the fA of Bn plantlets was the lowest when the nitrate concentration was 10 mM (Fig. 5). As a result, the Bn plantlets obtained the maximum CC/NN ratio when the nitrate concentration was 10 mM. Hence, an appropriate concentration of nitrate (10 mM) contributes to enhancing the C sink for Bn plantlets.

Under the condition that each treatment contained 20 mM ammonium, increasing the nitrate concentration contributed to elevating the proportion of assimilated nitrate for Bn plantlets when its concentration was in the range of 10 to 40 mM (Fig. 5). However, the maximum proportion of assimilated nitrate is only approximately 0.35 even if the concentration of nitrate is twice that of ammonium. These results indicate that the foliar nitrogen content of Bn plantlets is mainly derived from the assimilation of ammonium. The assimilation of ammonium is predominant among all treatments for Bn plantlets, which may be attributed to the lower energy cost for the assimilation of ammonium in comparison to the assimilation of nitrate [38, 39]. Generally, the assimilation of one nitrate molecule consumes 20 ATP, while the assimilation of one ammonium molecule only costs 5 ATP [38]. Consequently, the energy cost for the assimilation of 1 mol nitrogen will be in the range of 5 to 20 mol ATP for plantlets grown in a mixed N source. The proportion of assimilated nitrate reached the minimum value (approximately 0.2) for Bn plantlets when the nitrate concentration was 10 mM. As a result, we can conclude that the minimum energy cost of assimilating 1 mol nitrogen is approximately 8 mol ATP for Bn plantlets grown in the mixed N sources containing 20 mM ammonium. Hence, quantifying the proportion of assimilated nitrate and ammonium contributes to revealing the energy efficiency for N assimilation in plantlets grown in mixed N sources.

Plants usually suffer from ammonium toxicity when ammonium is supplied at high concentrations [2, 40]. However, ammonium toxicity can be alleviated by the addition of nitrate [1,2,3],or exogenous C sources [41, 42]. As shown in Table 1, under the condition that each treatment contained 20 mM ammonium, the increased biomass of Bn plantlets showed no significant change when the nitrate concentration was in the range of 5 to 20 mM. The Bn plantlets did not show apparent growth suppression when the nitrate concentration was only 5 mM, which might be related to the relatively high proportion of assimilated nitrate (Fig. 5). A recent study indicated that acidic stress caused by excessive ammonium assimilation is the primary cause of ammonium toxicity [43]. As shown in Fig. 1, the foliar nitrogen content of Bn plantlets was relatively high (above 5%) among all treatments. Hence, a relatively high proportion of assimilated nitrate contributes to alleviating acidic stress because the assimilation of nitrate is accompanied by the consumption of protons [43]. Moreover, an adequate supply of sucrose also led to the alleviation of ammonium toxicity in Bn plantlets [41, 42]. To some extent, increasing the nitrate concentration improves the growth of Bn plantlets. Therefore, C metabolism may be affected by the nitrate concentration in Bn plantlets.

The growth of Bn plantlets depended on the CO2 assimilation and sucrose utilization in this study. The proportion of assimilated CO2 can indicate the degree of photoautotrophy (i.e., the photosynthetic capacity) [7]. As shown in Fig. 3, the proportion of assimilated CO2 was obviously lower at the minimum nitrate concentration (5 mM) than at the other nitrate concentrations. The lowest proportion of CO2 assimilation indicates the weakest photosynthetic capacity. The poor photosynthetic capacity of Bn plantlets fed with the maximum ammonium/nitrate ratio (20 mM ammonium, 5 mM nitrate) may be attributed to the serious lack of chlorophyll because the low chlorophyll content in leaves usually limits the photosynthetic capacity of plants [44].

The chlorophyll content is usually positively correlated with the foliar nitrogen content because most leaf N is present in chloroplasts [45]. The foliar nitrogen content of Bn plantlets was relatively high (above 5%) and was almost the same when the nitrate concentration was in the range of 5 to 20 mM (Fig. 1). However, the chlorophyll content of Bn plantlets was obviously lower at 5 mM nitrate concentration than at the other nitrate concentrations (Table 2). The lowest chlorophyll content of Bn plantlets at 5 mM nitrate may be related to the acidic stress caused by excessive ammonium assimilation [43]. Acidic stress can cause a distinct decline in magnesium accumulation in plants [46]. Acidic stress can be significantly alleviated by an adequate supply of nitrate, whereas an insufficient supply of nitrate may not effectively alleviate acidic stress [47]. Hence, we speculate that the magnesium limit may inhibit the biosynthesis of chlorophyll when the nitrate concentration is only 5 mM.

With increasing nitrate concentration, the amount of nitrogen in leaves derived from nitrate assimilation (NAAnitrate) increases gradually (Fig. 6), which suggests that increasing the nitrate concentration enhances the assimilation of nitrate. Nitrate is mainly transported by NRTs, most of which are 2H+/1NO3 symporters [48, 49]. Hence, increasing the nitrate concentration can alleviate the acidic stress caused by excessive ammonium assimilation. As a result, the biosynthesis of chlorophyll is improved due to the increased nitrate concentration (Table 2). A previous study found that an increased chlorophyll concentration usually leads to an increase in energy and reducing power [50]. However, we did not find that the photosynthetic capacity of Bn plantlets showed a linear relationship with the chlorophyll concentration. As shown in Fig. 3, the photosynthetic capacity of Bn plantlets was nearly the same when the nitrate concentration were 10 mM and 20 mM. The photosynthetic capacity of Bn plantlets did not increase when the nitrate concentration increased from 10 to 20 mM, which may be attributed to the elevated proportion of assimilated nitrate (Fig. 5). The increased proportion of assimilated nitrate consumes more energy and reducing power. As a whole, the increase in chlorophyll concentration may contribute to improving the photosynthetic capacity.

It is widely known that futile ammonium cycling occurs when ammonium is supplied at high concentrations, which results in a large energy loss [51,52,53]. The degree of futile ammonium cycling may be determined by the nitrate concentration [1]. Our results show that the assimilation of ammonium is obviously promoted by increasing nitrate concentrations (Fig. 6). The enhanced assimilation of ammonium leads to the reduction of futile ammonium cycling. Generally, excessive ammonium assimilation can promote acidic stress, which is considered to be the primary cause of ammonium toxicity [43]. However, increasing the supply of nitrate not only promotes the assimilation of ammonium, but also enhances the assimilation of nitrate (Fig. 6). The proportion of assimilated ammonium gradually decreased for Bn plantlets when the nitrate concentration increased from 10 to 40 mM. The nitrate reduction is accompanied by a consumption of H+ and leads to the production of a hydroxyl ion [38]. Hence, the alleviation of ammonium toxicity may be attributed to the reduction of futile ammonium cycling and the relief of acidic stress by the nitrate reduction process.

The effective coordination of C and N metabolism contributes to the optimal growth of plants [54]. Hence, the growth of Bn plantlets is improved when the nitrate concentration reaches 10 mM, which may be attributed to the minimum energy cost used for N assimilation per mole, the reduction of futile ammonium cycling, and the elevated photosynthetic capacity. In general, increasing the nitrate concentration contributes to improving the growth of Bn plantlets. Based on the effective management of inorganic N supply, it is necessary to know the utilization coefficients of nitrate and ammonium for plantlets grown in the culture media. Hence, quantifying the utilization coefficients of nitrate and ammonium contributes to optimizing the inorganic N supply for the plantlets. As shown in Fig. 7, the utilization coefficient of nitrate did not increase with increasing nitrate concentration. Meanwhile, the utilization coefficient of ammonium did not show an obvious increase when the nitrate concentration increased from 10 to 20 mM (Fig. 7). Furthermore, the photosynthetic capacity of Bn plantlets also did not increase when the nitrate concentration increased from 10 to 20 mM. Hence, given a basal concentration of 20 mM ammonium, the supply of 10 mM nitrate was the optimal combined concentration to improve N use efficiency for Bn growth.

Conclusions

Based on the bidirectional stable nitrogen isotope tracer technique, the proportion of assimilated nitrate and ammonium can be quantified for Bn plantlets grown at variable ammonium/nitrate ratios. The minimum energy cost of assimilating 1 mol N is approximately 8 mol ATP for Bn plantlets grown in the mixed N sources containing 20 mM ammonium. The utilization proportion of sucrose and CO2 can be quantified by a two end-member isotope mixing model for Bn plantlets grown at variable ammonium/nitrate ratios. Quantifying the utilization proportions nitrate, ammonium, sucrose and CO2 contributes to revealing the difference in the ammonium/nitrate use efficiency for new C input derived from CO2 assimilation/sucrose utilization in plantlets grown at variable ammonium/nitrate ratios and provides a new insight that the nitrate-dependent alleviation of ammonium toxicity might be attributed to the stimulation of ammonium assimilation that would mitigate the futile ammonium cycling. We also postulate an enhancement of photosynthesis by nitrate and relief of acidic stress by the nitrate reduction process.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its Additional file 1: Table S1.

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Acknowledgements

The authors would like to thank the technical staff at the State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, for technical assistance during the measurement of the δ15N and δ13C, in particular Jing Tian and Ning An.

Funding

This work was supported by the National Key Research and development Program of China (2016YFC0502607), the National Key Research and Development Program of China (2021YFD1100300), and the Science and Technology Innovation Talent Project of Guizhou Province (No. (2016)5672).

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Y.Y. Wu and K.Y. Zhang conceived and designed the experiment. K.Y. Zhang performed most of the experiment. H.T. Li performed some of the experiment. K.Y. Zhang and Y. Su performed the analyses. K.Y. Zhang and Y.Y. Wu wrote the manuscript. The author(s) read and approved the final manuscript.

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Correspondence to Yanyou Wu.

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Additional file 1: Table S1

. The leaf dry weight of Brassica napus plantlets cultured under nitrate treatment.

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Zhang, K., Wu, Y., Su, Y. et al. Implication of quantifying nitrate utilization and CO2 assimilation of Brassica napus plantlets in vitro under variable ammonium/nitrate ratios. BMC Plant Biol 22, 392 (2022). https://doi.org/10.1186/s12870-022-03782-8

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Keywords

  • Ammonium
  • Bidirectional stable isotope tracer
  • Isotope mixing model
  • Nitrate
  • Nitrogen assimilation
  • Nitrogen use efficiency