the coupling of carbon and nitrogen in response to
TRANSCRIPT
The Coupling of Carbon and Nitrogen in Responseto Variable NH4+/ NO3- Ratios in Brassica NapusPlantlets in VitroKaiyan Zhang
Guizhou Normal UniversityYanyou Wu ( [email protected] )
Institute of Geochemistry Chinese Academy of Sciences https://orcid.org/0000-0002-0940-4349Yue Su
Guizhou Vocational College of AgricultureHaitao Li
Guizhou Vocational College of Agriculture
Research
Keywords: ammonium, bidirectional stable isotope tracer, carbon �xation, nitrate, nitrogen assimilation,quanti�cation, stable isotopes
Posted Date: September 30th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-934300/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
1
The coupling of carbon and nitrogen in response to variable NH4+/ NO3- ratios in 1
Brassica napus plantlets in vitro 2
3
Kaiyan Zhang1, Yanyou Wu2*, Yue Su3 and Haitao Li3 4
5
The work was carried out at State Key Laboratory of Environmental Geochemistry, 6
Institute of Geochemistry, Chinese Academy of Sciences. 7
Address: No. 99 Lincheng West Road, Guanshanhu District, Guiyang, Guizhou 8
Province 550081, P.R.China 9
Email: [email protected]; [email protected]; [email protected]; 10
Correspondence: 12
Yanyou Wu 13
Tel:+86 0851 8439 1746 14
2
The coupling of carbon and nitrogen in response to variable NH4+/ NO3- ratios in 15
Brassica napus plantlets in vitro 16
Abstract 17
Background: Plantlets grown in vitro with a mixed nitrogen source utilize sucrose and 18
CO2 as carbon sources for growth. However, it is very difficult to obtain the correct 19
proportion of assimilated nitrate, ammonium, sucrose and CO2 for plantlets. 20
Consequently, the NH4+/NO3
- use efficiency for carbon fixation derived from the 21
assimilation of sucrose/CO2 is still unclear for plantlets. 22
Results: The bidirectional stable nitrogen isotope tracer technique was employed to 23
quantify the proportions of nitrate and ammonium utilized at different NH4+/ NO3
- ratios, 24
and the proportions of sucrose and CO2 assimilation were quantified by the foliar δ13C 25
values of plantlets. There was an obvious difference in the assimilation of nitrate and 26
ammonium under different NH4+/NO3
- ratios for Brassica napus (Bn) plantlets. 27
Increasing the supply of nitrate contributed to enhancing the assimilation of nitrate and 28
ammonium simultaneously. The nitrate utilization coefficients of the Bn plantlets had 29
no distinct change with increasing nitrate concentration, while the ammonium 30
utilization coefficients of the Bn plantlets increased obviously with increasing nitrate 31
concentration. The proportion of sucrose/CO2 assimilation depended on the NH4+/NO3
- 32
ratios of the Bn plantlets. Both nitrate and ammonium assimilation were independent 33
of sucrose/CO2 assimilation. Based on the proportion of CO2, sucrose, nitrate and 34
ammonium utilization, the nitrate/ammonium use efficiency (as indicated by the C/N 35
ratio) for carbon fixation derived from the assimilation of sucrose/CO2 can be 36
quantified for Bn plantlets. 37
Conclusions: Quantifying the utilization proportions of nitrate and ammonium can 38
reveal the difference in nitrate and ammonium utilization among plantlets at different 39
3
NH4+/NO3
- ratios. Foliar δ13C value in combination of the foliar δ15N value of plantlets 40
can be used to quantify the nitrate/ammonium use efficiency for the carbon fixation 41
derived from the assimilation of sucrose/CO2, which contributes to knowing the 42
coupling process of carbon and nitrogen in plantlets and provides an alternate way to 43
optimize the supply of inorganic nitrogen in culture media. 44
Keywords: ammonium, bidirectional stable isotope tracer, carbon fixation, nitrate, 45
nitrogen assimilation, quantification, stable isotopes 46
47
Background 48
Nitrogen (N) is an essential nutrient for the growth and development of plants. 49
Generally, the growth and productivity of crops heavily depend on the N supply [1]. 50
Ammonium (NH4+) and nitrate (NO3
-) are the two main forms of nitrogen acquired by 51
plants under natural conditions. Compared to nitrate, ammonium is thought to be the 52
preferred N source for plants due to its low energy cost [2]. However, plants usually 53
show symptoms of ammonium toxicity when ammonium is supplied as the sole N 54
source or is present at millimolar concentrations [3]. In general, a combination of NH4+ 55
and NO3- could effectively alleviate ammonium toxicity [3, 4]. Most plants grow well 56
if both nitrate and ammonium are supplied simultaneously [5, 6]. Hence, it is necessary 57
to know the contribution of NH4+/ NO3
- to total inorganic nitrogen assimilation in plants, 58
which contributes to revealing the mechanism of inorganic nitrogen utilization. 59
Nitrate and ammonium are widely used in plant tissue culture. Generally, the growth 60
of plantlets in vitro is dependent on the total N amount and/or the proportion of NH4+ 61
and NO3- [7]. Nonoptimal N amounts and ratios of nitrate to ammonium may cause 62
stunted growth and physiological disorders in plantlets [8]. For some plant cultures, the 63
amount of inorganic N used in growth media far exceeds the amount required for 64
4
normal growth, which results in a waste of inorganic N. In addition, the concentration 65
of nitrate is usually higher than that of ammonium for most growth media [9]. From the 66
view of the energy cost of N assimilation, an excessive supply of nitrate is not 67
economical because the assimilation of nitrate consumes more energy and reductants 68
than the assimilation of ammonium [2]. Therefore, it is necessary to study the 69
proportion of nitrate and ammonium utilization by plantlets when both nitrate and 70
ammonium are supplied simultaneously, which may contribute to uncovering the 71
interactive mechanisms of nitrate and ammonium. 72
During the multiplication stage, most plantlets utilize sucrose and CO2 as carbon (C) 73
sources for growth [10]. Therefore, the carbon in plantlets was derived from the 74
assimilation of sucrose and CO2. The carbon isotopic composition (δ13C) of leaves is 75
derived from the mix of the δ13C values of assimilated sucrose and CO2. As a result, the 76
proportion of sucrose and CO2 utilization could be obtained when the carbon isotope 77
fractionation values of sucrose and CO2 assimilation are known. Quantifying the 78
proportion of CO2 utilization can contribute to evaluating the photosynthetic capacity 79
of plantlets. The survival rate of plantlets during acclimation is positively correlated 80
with their photosynthetic capacity [11]. However, the photosynthetic capacity of 81
plantlets is usually suppressed by high sucrose concentrations in culture medium [12]. 82
Hence, quantifying the proportion of CO2 utilization provides a new way to optimize 83
the growth of plantlets. Moreover, quantifying the proportion of sucrose and CO2 84
utilization can reveal the differential contribution of sucrose and CO2 to the total C 85
assimilation of plantlets. As a result, the mechanism of carbon assimilation in plantlets 86
can be revealed in detail. 87
Generally, C and N metabolism is tightly linked in plants [13]. The photosynthetic 88
capacity of leaves is closely related to the foliar N content because most leaf N is present 89
5
in chloroplasts. The reducing power and C skeletons provided for N assimilation are 90
derived from photosynthesis and sugar metabolism [14]. Therefore, photosynthetic 91
carbon assimilation and nitrate/ammonium assimilation are coupled in plants. 92
Accordingly, sucrose/CO2 assimilation and nitrate/ammonium assimilation are coupled 93
in plantlets. 94
The plant C/N ratio can be employed as a proxy measure of N-use efficiency (NUE) 95
over time [15]. Therefore, when the C and N contents in leaves are measured, different 96
leaf C/N ratios could be obtained for the plantlets if the proportions of CO2, sucrose, 97
nitrate and ammonium utilization are known. As a result, the nitrate/ammonium use 98
efficiency (as indicated by the C/N ratio) for carbon fixation derived from the 99
assimilation of sucrose/CO2 can be quantified. 100
Currently, it is difficult to obtain the assimilation of nitrate and ammonium by 101
measuring the consumption of inorganic N in the presence of agar. The optimization of 102
inorganic N supplies for plantlets is based on applying a series of different 103
concentrations of nitrate and ammonium [7, 8]. However, this approach is very 104
inefficient and not conducive to quantification of the proportion of nitrate and 105
ammonium utilization. Hence, there is a need for a high-efficiency method in which the 106
proportion of nitrate and ammonium utilization can be quantified. 107
Generally, the nitrogen isotope composition (δ15N) of plants is strongly connected to 108
the δ15N of the culture substrate [16, 17]. Therefore, plant δ15N is widely used as an 109
indicator of nitrogen sources [18, 19, 20]. In addition, the δ15N in plant tissue is used to 110
reveal the preference of plants for special N sources [21, 22]. Both nitrate reductase 111
(NR) and glutamine synthetase (GS) discriminate against 15N relative to 14N [15, 23]. 112
Hence, nitrogen isotope fractionation occurs during the assimilation of nitrate and 113
ammonium. The nitrogen isotope discrimination of NR is in the range of 19–22‰ [23, 114
6
24, 25] or 26‰ [26], whereas the nitrogen isotope fractionation value of GS is 16.5±1.5‰ 115
[27]. Moreover, compared to roots, shoots are often enriched in 15N regardless of the 116
inorganic nitrogen forms of NO3− or NH4
+ [28]. Therefore, N isotope fractionation 117
should be considered when the δ15N values of plants are used to study the characteristics 118
of inorganic N assimilation. 119
The assimilation of inorganic nitrogen occurs in the roots and/or shoots depending 120
on plant species and available N form [29, 30]. Discrimination factors for NR and GS 121
are obviously different. In addition, the δ15N values of different amino acids in leaves 122
show distinct differences [31]. As a result, it is very difficult to interpret the δ15N of 123
plants grown in mixed N sources. The nitrogen isotope fractionation values of nitrate 124
assimilation and ammonium assimilation are also difficult to obtain simultaneously. 125
Usually, because of N isotope fractionation during uptake, assimilation, translocation 126
and remobilization [32], quantifying the proportion of nitrate and ammonium utilization 127
is not possible with the δ15N of plants when a single isotope tracer is used at near-natural 128
abundance levels. However, the assimilation of nitrate and ammonium only occurred 129
in leaves in this study because the concentrations of cytokinin and auxin in this 130
experiment precluded root formation by the plantlets. As a result, the foliar δ15N values 131
of the root-free plantlets were only derived from the mix of the δ15N values of 132
assimilated nitrate and ammonium in leaves without interference from the assimilation 133
of nitrate and ammonium in the roots. Moreover, the cloned plantlets had no individual 134
differences and were maintained in the same culture conditions in this study. Hence, 135
based on the bidirectional stable N isotope tracer technique [33], the proportion of 136
nitrate and ammonium utilization can be quantified in root-free plantlets when two 137
labeled stable nitrogen isotope treatments (L- and H-labeled nitrate) are applied in this 138
study. 139
7
In the present study, plantlets of Brassica napus (Bn) were subjected to different 140
inorganic N regimes where the concentration of ammonium was set as 20 mM in each 141
treatment. The following were our main aims: (1) to reveal the differences in nitrate and 142
ammonium assimilation of Bn plantlets grown in variable NH4+/NO3
- ratios; (2) to 143
quantify the differential contributions of the NH4+/ NO3
- ratio to carbon fixation derived 144
from the assimilation of sucrose/CO2 in Bn plantlets; and (3) to reveal the coupled 145
relationship between the utilization of nitrate/ammonium and the assimilation of 146
sucrose/CO2 at different NH4+/ NO3
- ratios. 147
Methods 148
Plant materials and experimental treatments 149
Bn plantlets in vitro were employed as explants in this experiment. Single shoots of Bn 150
plantlets were grown in culture media with four inorganic nitrogen regimes. The 151
average fresh weight (FW) per shoot was 0.09 g for the Bn plantlets. Based on the 152
ammonium concentration (20 mM) in the MS culture medium, the ammonium 153
concentration was set as 20 mM in each treatment, and the nitrate concentrations in the 154
four treatments was set at 5 mM, 10 mM, 20 mM and 40 mM. Accordingly, the 155
ammonium:nitrate ratio was different in each treatment. Each inorganic nitrogen regime 156
included two labeled stable nitrogen isotope treatments. The labeled treatments were 157
separated into groups with high (H) and low (L) natural 15N-abundance in the NaNO3, 158
with a δ15N of 22.67‰ in H and of 8.08‰ in L. NH4Cl, with a δ15N of -2.64‰, was 159
employed as the ammonium nitrogen in this experiment. Each Erlenmeyer flask (150 160
ml) contained 50 ml Murashige and Skoog (MS) [34] medium supplemented with 2.0 161
mg·L-1 6-benzylaminopurine, 0.2 mg·L-1 α-naphthylacetic acid, 3% (w/v) sucrose, and 162
7.5 g·L-1 agar. The concentrations of cytokinin and auxin in this experiment precluded 163
root formation for Bn plantlets in vitro during the whole culturing stage. All culture 164
8
media were adjusted to pH 5.8 and then autoclaved at 121 °C for 20 min. The Bn 165
plantlets were maintained in a growth chamber with a 12-h photoperiod (50 μmol m-2 166
s-1 PPFD) at 25 ± 2 °C. 167
Determination of growth parameters 168
After 5 weeks of culturing, the Bn plantlets were removed from the Erlenmeyer flasks 169
in the afternoon. The biomass of each Bn plantlet (FW) was measured. Additionally, 170
the leaf biomass of each Bn plantlet was also measured. Next, the shoots of each Bn 171
plantlet were counted. Then, the leaves of the Bn plantlets were dried at 60 °C. 172
Moreover, the leaf dry weight (DW) of each Bn plantlet was also measured [see 173
Additional file 1]. Finally, the dried leaves were ground to a fine powder. 174
Chlorophyll concentration determination 175
A total of 0.1 g of fresh leaf that had been triturated in a mortar with a small amount of 176
liquid nitrogen was macerated with 10 ml 95% ethanol for 24 h at 4 °C. The chlorophyll 177
concentration in the extract was spectrophotometrically determined at 665 and 649 nm. 178
The concentrations, including chlorophyll a and chlorophyll b concentrations, were 179
determined on a fresh weight basis (mg·g–1) and calculated according to Alsaadawi et 180
al. [35]. 181
Analysis of elements and determination of δ15N and δ13C in plantlets 182
The total nitrogen and carbon contents of the dried leaves were determined using an 183
elemental analyzer (vario MACRO cube, Germany). Both δ15N and δ13C were 184
measured by a gas isotope ratio mass spectrometer (MAT-253, Germany). Isotope ratios 185
were calculated as: 186
13 15samples sample standardδ[ C, N] =( / 1) 1000R R (1) 187
where Rsample refers to the 13C/12C or 15N/14N of the plant material, and Rstandard refers to 188
the isotope ratio of a known standard (PDB or N2 in air). International isotope secondary 189
9
standards of known 13C/12C ratios (IAEA CH3 and IAEA CH6) were used for calibration 190
to a precision of 0.1‰. For nitrogen, isotope secondary standards of known 15N/14N 191
ratios (IAEA N1, IAEA N2, and IAEA NO3) were used to calibrate the instrument to 192
reach a precision of 0.2‰ [36]. 193
Quantification of the contributions of nitrate and ammonium to total inorganic 194
nitrogen assimilation 195
The proportions of nitrate and ammonium assimilated by Bn plantlets were determined 196
with the bidirectional stable nitrogen isotope tracer technique [33]. Thus, the proportion 197
of assimilated nitrate (fA) contributing to total inorganic nitrogen assimilation was 198
calculated as follows: 199
A T T A Aδ δ / δ δH L H L
f (2) 200
where δTH is the foliar δ15N value of the plantlets cultured with mixed-nitrogen sources, 201
whose δ15N of nitrate in culture media was 22.67‰. δTL is the foliar δ15N value of the 202
plantlets cultured with mixed-nitrogen sources, whose δ15N of nitrate in culture media 203
was 8.08‰. Accordingly, δAH and δAL are the δ15N values derived from nitrate 204
assimilation. The proportion of assimilated ammonium (fB) contributing to total 205
inorganic nitrogen assimilation was calculated using the following equation: 206
B A1f f (3) 207
The standard deviations (SDs) of fA and fB were achieved by the error propagation 208
formula. 209
In this study, δTH and δTL could be obtained directly. However, when the plantlets 210
were cultured in the medium with mixed-nitrogen sources, it would have been difficult 211
to directly obtain δAH and δAL, which were involved in nitrogen isotope discrimination 212
in nitrate assimilation and the exchange of unassimilated nitrate between the shoot and 213
the substrate during the whole culture period. Hence, δAL and δAH changed over time in 214
10
this experiment. However, we were able to obtain δAL and δAH when the plantlets were 215
grown in culture medium in which nitrate was the sole nitrogen source. 216
The δAL and δAH in NO3--fed plantlets could be affected by unassimilated nitrate. 217
However, a previous study found that the storage pool of nitrate in leaves of tomato and 218
tobacco plants were replenished in the dark and became depleted in the light, and the 219
foliar nitrate concentration of tomato and tobacco plants reached a low level in the 220
afternoon [37,38]. Hence, when the plantlets had been cultured for 5 weeks and 221
harvested in the afternoon, the amount of unassimilated nitrate in the leaves of plantlets 222
would be very small in comparison with the amount of assimilated nitrate. Moreover, 223
the foliar δ15N value of plantlets did not vary significantly among nitrate concentrations 224
ranging from 10 mM to 40 mM [39], which suggested that the effect of unassimilated 225
nitrate in leaves on the foliar δ15N value could be neglected. As a result, the δAL and δAH 226
of plantlets grown in mixed-nitrogen sources could be replaced by the δAL and δAH in 227
NO3--fed plantlets. 228
In this study, the foliar δ15N values of NO3--fed plantlets that had been cultured for 229
5 weeks could be regarded as the δ15N values (δAL or δAH) of plantlets cultured in the 230
medium with mixed-nitrogen sources. A previous study found that the foliar δ15N values 231
of plantlets did not vary significantly among nitrate concentrations in culture medium 232
ranging from 10 mM to 40 mM [33, 39]. Sodium nitrate with a δ15N of 22.67‰/8.08‰ 233
was used as the sole nitrogen source in their study. Hence, the average foliar δ15N value 234
in NO3--fed plantlets at the three nitrate supply levels (10, 20, and 40 mM) was 235
approximately equal to the δ15N value (δAL or δAH) of plantlets cultured in the medium 236
with mixed-nitrogen sources in this study. As a result, we were able to obtain δAL and 237
δAH. δAL was 3.17±0.35‰ (n=9) for the Bn plantlets [39], and δAH was 15.19±0.86‰ 238
(n=9) for the Bn plantlets [33]. After determining δTH, δTL, δAH and δAL, we were able to 239
11
calculate fA and fB. 240
Quantifying the contribution of NO3-/NH4+ utilization to the amount of nitrogen 241
in leaves 242
The nitrogen accumulation amount (NAA) of the leaves was the absolute nitrogen 243
content in the dried leaves, and was calculated using the following equation: 244
NAA DW N content (4) 245
where the N content of the dried leaves was determined by an elemental analyzer. 246
The nitrogen in leaves was derived from the assimilation of nitrate and ammonium. 247
Therefore, the contributions of assimilated nitrate/ammonium to the amount of nitrogen 248
in leaves could be calculated by the following equations: 249
nitrate ANAA NAA f (5) 250
ammonium BNAA NAA f (6) 251
where NAAnitrate is the amount of nitrogen in leaves derived from nitrate assimilation, 252
and NAAammonium is the amount of nitrogen in leaves derived from ammonium 253
assimilation. The standard deviation (SD) of NAAnitrate and NAAammonium was calculated 254
by the error propagation formula. 255
Nitrogen utilization coefficient (NUC) of ammonium and nitrate 256
The nitrogen utilization coefficient (NUC) is the ratio of the total nitrogen content in 257
the dried leaves relative to the nitrogen content in the medium. Therefore, the nitrogen 258
utilization coefficient of ammonium (NUCammonium) and nitrate (NUCnitrate) could be 259
calculated by the following equation: 260
ammonium ammonium ammoniumNUC % NAA / M / n 100 （7） 261
nitrate nitrate nitrateNUC % NAA / M / n 100 （8） 262
where M is the molar mass of nitrogen, and nammonium and nnitrate are the number of moles 263
12
of ammonium and nitrate in the medium, respectively. The standard deviation (SD) of 264
NUCammonium and NUCnitrate was calculated by the error propagation formula. 265
Quantifying the proportion of inorganic carbon utilization in Bn plantlets 266
In this study, the Bn plantlets had access to both inorganic carbon and organic carbon, 267
namely, CO2 and sucrose. Therefore, the foliar δ13C value of the Bn plantlet was derived 268
from the mix of the δ13C values of assimilated inorganic and organic carbon. Based on 269
a two end-member isotope mixing model, an equation representing this utilization of 270
two different carbon sources by Bn plantlets can be established as follows: 271
T P C P Sδ δ 1 δf f (9) 272
where δT is the foliar δ13C value of Bn plantlets grown in mixed-carbon sources, and 273
could be obtained directly. fP is the proportion of assimilated CO2. 1-fP is the proportion 274
of assimilated sucrose. δC is the δ13C value derived from CO2 assimilation. δS is the 275
δ13C value derived from sucrose assimilation. Accordingly, equation (9) can be 276
rewritten as equation (10) 277
P T S C Sδ δ / δ δf (10) 278
The standard deviation (SD) of fP was achieved by the error propagation formula. 279
When the plantlets were grown in mixed-carbon sources, it would have been difficult 280
to directly obtain δS and δC. To obtain δS, the Bn plantlets were cultured in a CO2-free 281
atmosphere where CO2 was absorbed by soda lime, and sucrose was the only carbon 282
source for Bn plantlets. As a result, the isotope fractionation value of sucrose 283
assimilation could be obtained indirectly. The isotope fractionation value of sucrose 284
assimilation was 2.54±0.23‰ (n=3) for Bn plantlets. 285
Bn plantlets cannot survive without a supply of sucrose. Hence, the isotopic 286
fractionation value of CO2 assimilation cannot be obtained directly for Bn plantlets. To 287
obtain δC, the seeds of Bn were grown in MS culture medium without sucrose. After 5 288
13
weeks of culturing, the leaves were harvested for the measurement of δ13C. The foliar 289
C content of the Bn plantlets, which were derived from germinated seeds, was only 290
derived from CO2 assimilation. Hence, the foliar δ13C value of the Bn plantlets, which 291
were derived from seed germination, could be used to approximate δC in this study. As 292
a result, δC could be obtained indirectly and was -28.05±0.22‰ (n=3) for Bn plantlets 293
in this study. After δT, δS and δC were known, we were able to calculate fP. 294
The C/N ratios of leaves 295
After determining the carbon (CT) and nitrogen (NT) contents of leaves, the CT/NT ratio 296
of leaves could be obtained directly. In this study, the proportions of nitrate, ammonium, 297
CO2 and sucrose assimilation were calculated for Bn plantlets. Accordingly, the carbon 298
content derived from the assimilation of CO2 (CA) and sucrose (CH) could be obtained; 299
the nitrogen content derived from the assimilation of nitrate (NN) and ammonium (NA) 300
could also be obtained. As a result, the nitrate use efficiency for carbon fixation (CA/NN 301
ratio), which is derived from the assimilation of CO2, the nitrate use efficiency for 302
carbon fixation (CH/NN ratio), which is derived from the assimilation of sucrose, the 303
ammonium use efficiency for carbon fixation (CA/NA ratio), which is derived from the 304
assimilation of CO2, and the ammonium use efficiency for carbon fixation (CH/NA ratio), 305
which is derived from the assimilation of sucrose can be calculated by the following 306
equations: 307
A N P T A T )C / N ratio ( C / () Nf f (11) 308
H N P T A T )C N )/ N ratio ((1 C ) / (f f (12) 309
A A P T A T )C / N ratio ( C / )((1 N)f f (13) 310
H A P T A T ) ) ) )C / N ratio ((1 C / ((1 Nf f (14) 311
The standard deviation (SD) of the CA/NN ratio, CA/NA ratio, CH/NN ratio, and CH/NA 312
ratio was achieved by the error propagation formula. 313
14
Statistical analysis 314
The data were subjected to analysis of variance (ANOVA). The means of the different 315
groups were compared via Tukey’s test (p<0.05). The data are shown as the mean ± 316
standard deviation (SD). 317
Results 318
Growth 319
The nitrate concentration had a significant effect on the growth of Bn plantlets. As 320
shown in Table 1, when the ammonium concentration remained 20 mM in each 321
treatment, increasing the supply of nitrate could obviously promote the growth of Bn 322
plantlets. In addition, the leaf biomass of Bn plantlets increased significantly with 323
increasing nitrate supply. With respect to the proliferation of shoots, the Bn plantlets 324
showed no significant difference with increasing nitrate concentration, except at the 325
lowest concentrations. Generally, Bn plantlets had good performance with respect to 326
shoot proliferation under all treatments (Table 1). 327
328
Chlorophyll concentrations 329
The chlorophyll concentration of the Bn plantlets was significantly affected by the 330
nitrate supply. Under the condition that each treatment included 20 mM ammonium, 331
the chlorophyll concentration of the Bn plantlets showed a positive response to 332
increasing nitrate concentrations. Increasing the supply of nitrate could promote the 333
biosynthesis of chlorophyll for Bn plantlets (Table 2). 334
335
Elemental analysis of the Bn plantlets 336
The leaf nitrogen content of Bn plantlets was relatively high in all treatments. Because 337
each treatment contained 20 mM ammonium, specific supplies of nitrate had no 338
15
obvious effect on nitrogen accumulation in Bn plantlets. As shown in Fig. 1, the leaf 339
nitrogen content of Bn plantlets was not significantly different when the nitrate supply 340
ranged from 5 mM to 20 mM. Moreover, the leaf carbon content of Bn plantlets did 341
not show a significant difference with increasing nitrate (Fig. 1). 342
343
Foliar carbon isotope ratio of the Bn plantlets 344
The effect of nitrate on the δ13C values of the Bn plantlets depended on the 345
concentration of nitrate in this study. The δ13C values of Bn plantlets showed no 346
significant difference with increasing nitrate concentrations, except at the lowest 347
concentrations. Generally, under the condition that each treatment included 20 mM 348
ammonium, the δ13C values of the Bn plantlets gradually decreased with increasing 349
nitrate concentrations. The maximum and minimum δ13C values of Bn plantlets 350
occurred at 5 mM and 40 mM nitrate, respectively (Fig. 2). 351
352
The proportion of CO2 and sucrose utilization by the Bn plantlets 353
Under the condition that each treatment contained 20 mM ammonium, increasing the 354
supply of nitrate contributed to enhancing the proportion of CO2 utilization by Bn 355
plantlets (Fig. 3). However, when the supply of nitrate reached a certain level, the 356
proportion of CO2 utilization did not show an obvious increase with increasing nitrate 357
supply. Generally, the foliar carbon content of Bn plantlets is mainly derived from the 358
utilization of sucrose. 359
360
Foliar nitrogen isotope ratio of the Bn plantlets 361
The δ15N values of Bn plantlets cultured in the H and L treatments were very different 362
at all levels of nitrate supply. The δ15N values of the Bn plantlets were higher in the H 363
treatment than in the L treatment. The δ15N value of Bn plantlets was significantly 364
16
affected by nitrate concentration in both the H and L treatments. The maximum and 365
minimum δ15N values of Bn plantlets occurred at 40 mM and 5 mM nitrate, respectively 366
(Fig. 4). 367
368
The contribution of nitrate/ammonium to total inorganic nitrogen assimilation 369
Under the condition that each treatment contained 20 mM ammonium, the 370
nitrate/ammonium ratio gradually increased with increasing nitrate concentration. 371
However, the contribution of nitrate utilization to total inorganic nitrogen assimilation 372
did not show an obvious increase when the nitrate supply ranged from 5 mM to 20 mM 373
for the Bn plantlets. The contribution of nitrate utilization to total inorganic nitrogen 374
assimilation was distinctly lower than that of ammonium in all treatments. Even if the 375
concentration of nitrate was twice that of ammonium, ammonium was the primary 376
source of nitrogen that was assimilated by the Bn plantlets (Fig. 5). In general, 377
ammonium was preferred by the Bn plantlets. 378
379
The contribution of NO3-/NH4+ utilization to the amount of nitrogen in leaves 380
The amount of nitrogen in leaves (NAA) of Bn plantlets showed positive response to 381
increasing nitrate concentrations when each treatment contained 20 mM ammonium. 382
However, the increase in the NAA in Bn plantlets depended on the increase in nitrate 383
and ammonium assimilation. As shown in Fig. 6, with increasing nitrate concentration, 384
the amount of nitrogen in leaves derived from nitrate assimilation (NAAnitrate) gradually 385
increased, and the amount of nitrogen in leaves derived from ammonium assimilation 386
(NAAammonium) also increased. Increasing the supply of nitrate could simultaneously 387
promote the assimilation of nitrate and ammonium. Generally, NAAammonium was higher 388
than NAAnitrate in each treatment (Fig. 6). Therefore, the nitrogen in the leaves of Bn 389
17
plantlets is mainly derived from ammonium assimilation. The contribution of NO3-
390
utilization to the amount of nitrogen in leaves was lower than that of NH4+ utilization. 391
392
The utilization coefficients of nitrate and ammonium of the Bn plantlets 393
The utilization coefficients of nitrate and ammonium of the Bn plantlets showed 394
different responses to increasing nitrate concentrations when each treatment contained 395
20 mM ammonium. The nitrate utilization coefficients of the Bn plantlets had no 396
distinct change with increasing nitrate concentration, while the ammonium utilization 397
coefficients of the Bn plantlets increased obviously with increasing nitrate 398
concentration (Fig. 7). Increasing the supply of nitrate could not improve the nitrate 399
utilization coefficients of the Bn plantlets, but clearly enhanced the ammonium 400
utilization coefficients of the Bn plantlets. 401
402
The C/N ratios of leaves 403
There were clear differences between the CT/NT ratio, CA/NN ratio, CA/NA ratio, CH/NN 404
ratio, and CH/NA ratio of leaves in Bn plantlets (Table 3). Under the condition that each 405
treatment contained 20 mM ammonium, the CT/NT ratio of leaves had no obvious 406
change with increasing nitrate concentration, except at 120 mM nitrate. Generally, the 407
CH/NN ratio and CH/NA ratio of leaves decreased with increasing nitrate concentration. 408
With increasing nitrate concentration, the CA/NN ratio of leaves first increased and then 409
decreased, while the CA/NA ratio of leaves slowly increased. As shown in Table 3, the 410
CH/NN ratio of leaves was higher than the CH/NA ratio of leaves under each treatment; 411
the CA/NN ratio of leaves was also higher than the CA/NA ratio of leaves under each 412
treatment. Therefore, to some extent, the nitrate use efficiency was higher than that of 413
ammonium. Moreover, the CA/NN ratio of leaves was lower than the CH/NN ratio of 414
18
leaves under each treatment; the CA/NA ratio of leaves was also lower than the CH/NA 415
ratio of leaves under each treatment. Accordingly, the NN/CA ratio of leaves was higher 416
than the NN/CH ratio of leaves under each treatment; the NA/CA ratio of leaves was also 417
higher than the NA/CH ratio of leaves under each treatment. 418
419
Discussion 420
Plant δ15N represents an indicator of nitrogen sources [18, 19, 20]. Kalcsits et al [21] 421
found that the δ15N value in NO3--fed plants was very different from that in NH4
+-fed 422
plants. In this study, the δ15N values of the Bn plantlets showed large differences 423
between the L- and H-labeled treatments (Fig. 4). The foliar δ15N value of Bn plantlets 424
was derived from the mix of the δ15N values of assimilated nitrate and ammonium in 425
the leaves because no root formation occurred in this experiment. In addition, the δ15N 426
values of Bn plantlets in each treatment were different from those of the substrate, 427
which suggested that nitrogen isotope fractionation occurred during the assimilation of 428
inorganic nitrogen in the Bn plantlets. Generally, both the efflux of nitrate and 429
ammonium to the external media and the assimilation of nitrate and ammonium could 430
affect nitrogen isotope discrimination [40]. Therefore, if we were able to obtain the 431
nitrogen isotope fractionation values of assimilated nitrate and ammonium, it would be 432
possible to quantify the contribution of assimilated nitrate/ammonium to total inorganic 433
nitrogen assimilation with the δ15N values of the root-free plantlets in the L- or H-434
labeled treatments. However, it is very difficult to simultaneously obtain the nitrogen 435
isotope fractionation values of nitrate assimilation and ammonium assimilation when 436
the plantlets are grown in a mixed-nitrogen source. 437
According to the bidirectional stable nitrogen isotope tracer technique [33], when 438
two labeled stable nitrogen isotope treatments were used, it was unnecessary to 439
19
simultaneously obtain the nitrogen isotope fractionation values of nitrate assimilation 440
and ammonium assimilation. As shown in equation (2), the contribution of assimilated 441
nitrate to total inorganic nitrogen assimilation depended only on δTH, δTL, δAL and δAH. 442
δTH and δTL were the foliar δ15N values of the Bn plantlets grown in the mixed-nitrogen 443
source and could be obtained directly. δAL and δAH could be replaced by the foliar δ15N 444
values of the plantlets grown in the corresponding culture medium in which nitrate was 445
the sole nitrogen source. Hence, when δTH, δTL, δAH and δAL were determined, the 446
contribution of assimilated nitrate/ammonium to total inorganic nitrogen assimilation 447
could be quantified. 448
As shown in Fig. 5, although each treatment contained 20 mM ammonium, the 449
proportion of ammonium utilization by Bn plantlets was different among all treatments. 450
Generally, ammonium was the major source of nitrogen assimilated by the Bn plantlets 451
in all treatments. Even though the concentration of nitrate was twice that of ammonium, 452
the proportion of assimilated nitrate was far less than the proportion of assimilated 453
ammonium. Compared with nitrate, ammonium assimilation was predominant for Bn 454
plantlets because less energy is required [2]. Hence, to some extent, a sufficient supply 455
of nitrate might not be optimal for the effective management of the inorganic nitrogen 456
supply. 457
Nitrogen accumulation in leaves could represent the nitrogen acquisition capacity 458
of plants. In this study, we found that the nitrogen acquisition capacity of Bn plantlets 459
depended on the nitrate supply. As shown in Fig. 6, increasing the nitrate supply 460
promoted foliar nitrogen accumulation in Bn plantlets when each treatment contained 461
20 mM ammonium. However, the nitrate supply only contributed a small part of the 462
nitrogen accumulation in leaves. The increased nitrogen accumulation in the leaves of 463
Bn plantlets was mainly derived from ammonium assimilation. Generally, increasing 464
20
the nitrate supply could enhance the ability of Bn plantlets to acquire both nitrate and 465
ammonium. 466
Although an adequate nitrate supply could enhance the nitrogen acquisition 467
capacity of plants, an adequate nitrate supply was always accompanied by a low nitrate 468
utilization coefficient. As shown in Fig. 7, the utilization coefficients of nitrate did not 469
increase obviously with increased nitrate concentration, which suggests that excessive 470
nitrate application is a waste of nitrogen fertilizer. However, increasing the nitrate 471
supply contributed to the effective utilization of ammonium. Therefore, the utilization 472
coefficients of nitrate and ammonium should be taken into consideration together to 473
optimize the inorganic nitrogen supply. 474
In this study, although the foliar N content was mainly derived from ammonium 475
assimilation and was relatively high in each treatment containing 20 mM ammonium, 476
the utilization coefficient of ammonium differed obviously in each treatment. The 477
lowest utilization coefficient of ammonium occurred when nitrate was supplied at 5 478
mM, which suggested that Bn plantlets might suffer from ammonium toxicity. In 479
general, plants usually exhibit symptoms of ammonium toxicity when a high 480
concentration of ammonium is supplied [4]. Poor growth is often observed in plants 481
suffering from ammonium toxicity [41]. As shown in Table 1, the biomass of Bn 482
plantlets was minimal when nitrate was supplied at 5 mM. Therefore, the alleviation of 483
the adverse effect of high concentrations of ammonium depended on the amount of 484
nitrate supply. 485
The growth of Bn plantlets depended on the assimilation of sucrose and CO2 in 486
this study. Hence, the foliar δ13C value of Bn plantlets was derived from the mix of the 487
δ13C values of assimilated sucrose and CO2. According to equation (10), the proportion 488
of assimilated CO2 was determined for Bn plantlets. Subsequently, the proportion of 489
21
assimilated sucrose was determined for Bn plantlets. As shown in Fig. 3, the proportion 490
of assimilated CO2 was obviously lower at the minimum nitrate concentration (5 mM) 491
than at other nitrate concentrations. The lowest proportion of CO2 assimilation meant 492
the weakest photosynthetic capacity, which might have been caused by ammonium 493
toxicity [42]. The biosynthesis of chlorophyll is usually inhibited by ammonium 494
toxicity. As shown in Table 2, the chlorophyll content of leaves in Bn plantlets was the 495
lowest when nitrate was supplied at 5 mM. A low chlorophyll content in leaves usually 496
limits the photosynthetic capacity of plants [43]. Generally, the proportion of 497
assimilated sucrose/CO2 depended on the amount of nitrate supplied when each 498
treatment contained 20 mM ammonium. However, the proportion of NH4+/NO3
- 499
utilization did not show a positive response to the proportion of assimilated 500
sucrose/CO2 for Bn plantlets, which suggested that both nitrate assimilation and 501
ammonium assimilation were independent of the assimilation of sucrose/CO2. 502
Based on the effective management of inorganic N supply, enhancing the N use 503
efficiency contributed to reducing the supply of inorganic N. According to the 504
proportion of the assimilation of sucrose and CO2, we were able to obtain the foliar 505
carbon content derived from the assimilation of CO2 and sucrose, respectively. The 506
foliar N content derived from the assimilation of nitrate/ammonium could be obtained 507
by the proportion of nitrate and ammonium utilization. As a result, the 508
nitrate/ammonium use efficiency for carbon fixation derived from the assimilation of 509
sucrose/CO2 could be obtained. As shown in Table 3, the nitrate/ammonium use 510
efficiency (as indicated by the C/N ratio) for carbon fixation derived from the 511
assimilation of sucrose/CO2 differed obviously at different NH4+/ NO3
- ratios, which 512
suggested that the NH4+/ NO3
- ratio could affect the efficiency of carbon fixation 513
derived from the assimilation of sucrose/CO2. High use efficiency of nitrate and 514
22
ammonium for carbon fixation derived from the assimilation of sucrose and CO2 was 515
beneficial for the plantlets because the waste of inorganic N was avoided. Generally, it 516
was not ideal for plantlets to obtain the maximum nitrate and ammonium use efficiency 517
for carbon fixation derived from the assimilation of sucrose. The maximum nitrate and 518
ammonium use efficiency for carbon fixation derived from the assimilation of sucrose 519
usually meant poor photosynthetic capacity, which was detrimental to the survival of 520
plantlets during acclimatization [44]. Acquiring the maximum nitrate and ammonium 521
use efficiency for carbon fixation derived from the assimilation of CO2 contributed to 522
enhancing the carbon sink for plantlets. In this study, when each treatment contained 523
20 mM ammonium, there was an optimal concentration of nitrate for Bn plantlets to 524
realize the maximum nitrate use efficiency for carbon fixation derived from the 525
assimilation of CO2, which might be related to the fact that a low concentration of 526
nitrate could not effectively alleviate the ammonium toxicity and a high concentration 527
of nitrate was usually accompanied by low nitrate use efficiency [39]. The ammonium 528
use efficiency for carbon fixation derived from both sucrose assimilation and CO2 529
assimilation was relatively low for Bn plantlets in all treatments, which suggested that 530
the supply of ammonium might have well exceeded the amount required for the normal 531
growth of Bn plantlets. In addition, we found that the nitrate and ammonium 532
assimilation efficiency of Bn plantlets were higher at autotrophic growth than at 533
heterotrophic growth (Table 3), which demonstrate that photosynthesis provides the 534
additional reducing power for the assimilation of nitrate and ammonium. 535
Conclusions 536
Based on the bidirectional stable nitrogen isotope tracer technique, the proportion of 537
nitrate and ammonium utilization could be quantified for Bn plantlets. When each 538
treatment contained 20 mM ammonium, the foliar N of Bn plantlets was mainly derived 539
23
from the assimilation of ammonium, independent of the concentration of nitrate. The 540
ability of the Bn plantlets to acquire both nitrate and ammonium depended on the nitrate 541
supply. Increasing the supply did not improve the utilization coefficient of nitrate but 542
obviously enhanced the utilization coefficient of ammonium. The NH4+/ NO3
- ratio had 543
an obvious effect on the nitrate/ammonium use efficiency for carbon fixation derived 544
from the assimilation of sucrose/CO2. The difference in the nitrate/ammonium use 545
efficiency for carbon fixation derived from the assimilation of sucrose/CO2 at different 546
NH4+/ NO3
- ratios could be revealed by the foliar δ13C and δ15N values of plantlets. 547
Acknowledgements 548
The authors would like to thank the technical staff at the State Key Laboratory of 549
Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, 550
for technical assistance during the measurement of the δ15N and δ13C, in particular Jing 551
Tian and Ning An. 552
Authors’ Contributions 553
Y.Y. Wu and K.Y. Zhang conceived and designed the experiment. K.Y. Zhang 554
performed most of the experiment. H.T. Li performed some of the experiment. K.Y. 555
Zhang and Y. Su performed the analyses. K.Y. Zhang and Y.Y. Wu wrote the manuscript. 556
Funding 557
This work was supported by the National Key Research and development Program of 558
China (2016YFC0502607), the National Key Research and Development Program of 559
China (2021YFD1100300), and the Science and Technology Innovation Talent Project 560
of Guizhou Province (No. (2016)5672). 561
Availability of data and materials 562
All data generated or analyzed during this study are included in this published article 563
24
and its Additional file 1: Table S1. 564
Ethics approval and consent to participate 565
Not applicable. 566
Competing interests 567
The authors declare that they have no conflict of interest. 568
Consent for publication 569
Not applicable. 570
Author details 571
1School of Karst Science, Guizhou Normal University/State Engineering Technology 572
Institute for Karst Desertification Control, Guiyang 550001, China; 2State Key 573
Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese 574
Academy of Sciences, Guiyang 550081, China; 3Department of Agricultural 575
Engineering, Guizhou Vocational College of Agriculture, Qingzhen 551400, China. 576
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30
Table legends 701
Table 1 The growth parameters of Bn plantlets cultured under nitrate treatment. Note: 702
Bn-Brassica napus. Each nitrate treatment contained 20 mM ammonium. Each value 703
represents the mean ± SD (n=3). Values signed with the same letter in each line are not 704
significantly different by Tukey’s test (p>0.05). 705
Table 2 The chlorophyll concentration of Bn plantlets cultured under nitrate treatment. 706
Note: Bn-Brassica napus. Each nitrate treatment contained 20 mM ammonium. Each 707
value represents the mean ± SD (n=3). Values signed with the same letter in each line 708
are not significantly different by Tukey’s test (p>0.05). 709
Table 3 The C/N ratios of leaves in Bn plantlets cultured under nitrate treatment. Note: 710
Bn-Brassica napus. Each nitrate treatment contained 20 mM ammonium. Each value 711
represents the mean ± SD (n=3). The standard deviations of the CA/NN ratio, CA/NA 712
ratio, CH/NN ratio, and CH/NA ratio were calculated by the error propagation formula. 713
714
31
Figure legends 715
Fig. 1 Nitrogen content (a) and carbon content (b) the Bn plantlets cultured under nitrate 716
treatment. Bn Brassica napus. Each nitrate treatment contained 20 mM ammonium. 717
The nitrogen and carbon content was expressed as a percent of foliar dry weight, 718
respectively. The mean ± SD (n = 3) followed by different letters in the same legend 719
differ significantly (Tukey’s test, p < 0.05). 720
Fig. 2 The δ13C values of Bn plantlets in vitro cultured under nitrate treatment. 721
Note: Bn Brassica napus. Each nitrate treatment contained 20mM ammonium. The 722
mean ± SD (n=3) followed by different letters in the bar graph differ significantly 723
(Tukey’s test, p<0.05) 724
Fig. 3 The proportion of CO2 (a) and sucrose (b) utilization by the Bn plantlets cultured 725
under nitrate treatment. Note: Bn Brassica napus. Each nitrate treatment contained 20 726
mM ammonium. The error bars were calculated by the error propagation formula. 727
Fig. 4 The foliar δ15N values of the Bn plantlets cultured under nitrate treatment. Note: 728
Bn Brassica napus. Each nitrate treatment contained 20 mM ammonium. The mean ± 729
SD (n=3) followed by different letters in the same legend differ significantly (Tukey’s 730
test, p<0.05). 731
Fig. 5 The contribution of nitrate (a) and ammonium utilization (b) to total inorganic 732
nitrogen assimilation in the Bn plantlets cultured under nitrate treatment. Note: Bn 733
Brassica napus. Each nitrate treatment contained 20 mM ammonium. The error bars 734
were calculated by the error propagation formula. 735
Fig. 6 The amount of nitrogen in leaves (NAA), the amount of nitrogen in leaves 736
derived from nitrate assimilation (NAAnitrate), and the amount of nitrogen in leaves 737
derived from ammonium assimilation (NAAammonium) in the Bn plantlets cultured 738
under nitrate treatment. Note: Bn Brassica napus. Each nitrate treatment contained 20 739
32
mM ammonium. The error bars of NAAnitrate and NAAammonium were calculated by 740
the error propagation formula. 741
Fig. 7 The utilization coefficients of nitrate (a) and ammonium (b) in the Bn plantlets 742
cultured under nitrate treatment. Note: Bn Brassica napus. Each nitrate treatment 743
contained 20 mM ammonium. The error bars were calculated by the error propagation 744
formula. 745
746
33
Additional file 747
Table S1 The leaf dry weight of Bn plantlets cultured under nitrate treatment. Note: Bn-748
Brassica napus. Each nitrate treatment contained 20 mM ammonium. Each value 749
represents the mean ± SD (n=3). Values signed with the same letter in each line are not 750
significantly different by Tukey’s test (p>0.05). 751
752
Figures
Figure 1
Nitrogen content (a) and carbon content (b) the Bn plantlets cultured under nitrate treatment. Bn Brassicanapus. Each nitrate treatment contained 20 mM ammonium. The nitrogen and carbon content wasexpressed as a percent of foliar dry weight, respectively. The mean ± SD (n = 3) followed by differentletters in the same legend differ signi�cantly (Tukey’s test, p < 0.05).
Figure 2
The δ13C values of Bn plantlets in vitro cultured under nitrate treatment. Note: Bn Brassica napus. Eachnitrate treatment contained 20mM ammonium. The mean ± SD (n=3) followed by different letters in thebar graph differ signi�cantly (Tukey’s test, p<0.05)
Figure 3
The proportion of CO2 (a) and sucrose (b) utilization by the Bn plantlets cultured under nitrate treatment.Note: Bn Brassica napus. Each nitrate treatment contained 20 mM ammonium. The error bars werecalculated by the error propagation formula.
Figure 4
The foliar δ15N values of the Bn plantlets cultured under nitrate treatment. Note: Bn Brassica napus.Each nitrate treatment contained 20 mM ammonium. The mean ± SD (n=3) followed by different lettersin the same legend differ signi�cantly (Tukey’s test, p<0.05).
Figure 5
The contribution of nitrate (a) and ammonium utilization (b) to total inorganic nitrogen assimilation inthe Bn plantlets cultured under nitrate treatment. Note: Bn Brassica napus. Each nitrate treatmentcontained 20 mM ammonium. The error bars were calculated by the error propagation formula.
Figure 6
The amount of nitrogen in leaves (NAA), the amount of nitrogen in leaves derived from nitrateassimilation (NAAnitrate), and the amount of nitrogen in leaves derived from ammonium assimilation(NAAammonium) in the Bn plantlets cultured under nitrate treatment. Note: Bn Brassica napus. Eachnitrate treatment contained 20 mM ammonium. The error bars of NAAnitrate and NAAammonium werecalculated by the error propagation formula.
Figure 7
The utilization coe�cients of nitrate (a) and ammonium (b) in the Bn plantlets cultured under nitratetreatment. Note: Bn Brassica napus. Each nitrate treatment contained 20 mM ammonium. The error barswere calculated by the error propagation formula.
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