Nitrate and ammonium differ in their impact on δ
13C of plant metabolites and respired CO
2from tobacco leaves
Shiva Ghiasi1*, Marco M. Lehmann2, Franz-W. Badeck3, Jaleh Ghashghaie4, Robert Hänsch5,6, Rieke Meinen5, Sebastian Streb7, Meike Hüdig8, Michael E. Ruckle1, Dániel
Á. Carrera1, Rolf T. W. Siegwolf1,2, Nina Buchmann1, Roland A. Werner1
1Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland, 2Forest dynamics, Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland, 3Council for Agricultural Research and Economics, Research Centre for Genomics and Bioinformatics (CREA-GB), Fiorenzuola d´Arda, Italy, 4Laboratoire d'Ecologie, Systématique et Evolution (ESE), Université Paris-Sud, Orsay, France, 5Institute of Plant Biology, Technische Universität Braunschweig, Braunschweig, Germany,
6Center of Molecular Ecophysiology (CMEP), College of Resources and Environment, Southwest University, Chongqing, P.R. China, 7Department of Biology, ETH Zürich, Zürich, Switzerland, 8Institute of Molecular Physiology and Biotechnology of Plants (IMBIO), University of Bonn, Bonn, Germany
*Corresponding author: Shiva Ghiasi Email: shiva.ghiasi@usys.ethz.ch Phone: +41 78 952 5224
This document is the accepted manuscript version of the following article:
Ghiasi, S., Lehmann, M. M., Badeck, F. W., Ghashghaie, J., Hänsch, R., Meinen, R., … Werner, R. A. (2021). Nitrate and ammonium differ in their impact on δ13C of plant metabolites and respired CO2 from tobacco leaves.
Isotopes in Environmental and Health Studies, 57(1), 11-34.
https://doi.org/10.1080/10256016.2020.1810683
Abstract
The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant 1
performance. However, the effect of NO3- vs. NH4+ supply on δ13C of leaf metabolites and 2
respired CO2 is largely unknown. We supplied tobacco plants with a gradient of NO3- to 3
NH4+ concentration ratios and determined gas exchange variables, concentrations and δ13C of 4
TCA cycle intermediates, δ13C of dark respired CO2, and activities of key enzymes nitrate 5
reductase, malic enzyme and phosphoenolpyruvate carboxylase.
6
Net assimilation rate, dry biomass and concentrations of organic acids and starch decreased 7
along the gradient. In contrast, respiration rates, concentrations of intercellular CO2, soluble 8
sugars and amino acids increased. As NO3- decreased, activities of all measured enzymes 9
decreased. δ13C of CO2 and organic acids closely co-varied and were more positive under 10
NO3- supply, suggesting organic acids as potential substrates for respiration. Together with 11
estimates of intra-molecular 13C enrichment in malate, we conclude that a change in the 12
anaplerotic reaction of TCA cycle possibly contributes to 13C enrichment in organic acids and 13
respired CO2 under NO3- supply. Thus, the effect of NO3- vs. NH4+ on δ13C is highly relevant, 14
particularly if δ13C of leaf metabolites or respiration is used as proxy for plant performance.
15 16
Key words: ammonium (NH4+), nitrate (NO3-), nitrate reductase (NR), phosphoenolpyruvate 17
carboxylase (PEPC), compound specific carbon isotope analysis (CSIA), stable carbon 18
isotopes.
19 20
Introduction 21
The strong link between carbon (C) and nitrogen (N) within the plant-soil system has been 22
the subject of numerous studies [1–7]. Studying this interaction and its underlying 23
mechanisms has been critical for understanding the biochemical cycling of these elements, 24
particularly during respiration where C skeletons are provided for N assimilation [8]. Nitrate 25
(NO3-) and ammonium (NH4+) are the most important inorganic N species available to plants 26
[9], both following different uptake and assimilation patterns and resulting in fundamentally 27
different plant metabolism. Yet, the potentially different effects of these two inorganic N 28
species on the C isotopic composition (δ13C) of plant metabolites and dark respired CO2 are 29
largely unknown. This is particularly surprising, since increasing atmospheric CO2
30
concentrations have been found to decrease NO3- assimilation [10]. Therefore, a shift to NH4+
31
as a preferable source of N for plants might be expected in the future [11]. Equally important, 32
δ13C values of bulk leaves and leaf metabolites are widely used to study water use efficiency 33
(WUE), plant physiological processes and plant functional responses to climate. If nitrogen 34
assimilation has been or will be changing over time, the proxy of δ13C for WUE might be 35
unreliable. Thus, a better understanding of how different N species availability affects plant C 36
isotope fractionations is urgently needed.
37
NH4+ is described as a paradoxical nutrient because its redox state requires no reduction 38
process prior to its assimilation (via the GS-GOGAT pathway), but most plants with NH4+
39
supply grow slower and produce less biomass compared to those supplied with NO3- [12,13].
40
Former studies have shown physiological toxicity effects caused by NH4+ in plants [14–16], a 41
decrease in carbon assimilation rates [17], changes in the charge balance caused by reduced 42
uptake of cations, such as K+, Mg2+ and Ca2+, and an increased uptake of anions, such as 43
SO42-, PO43- and Cl- [12,18]. Moreover, NH4+ assimilation generates H+ ions which cause a 44
decrease in pH of the cytosol and eventually might result in growth depression [18].
45
Therefore, NH4+ assimilation takes place almost entirely in the roots [19], fuelled with C 46
skeletons and energy delivered from the leaves [2]. In contrast to NH4+ supply, about 90% of 47
the NO3- ions taken up by the roots are transported to the leaves for assimilation [20] together 48
with K+ as counter-ion [21]. NO3- assimilation involves reduction of NO3- to NH4+ by nitrate 49
and nitrite reductase (NR, NiR) enzymesprior to assimilation via the GS-GOGAT pathway 50
[22]. This reduction is coupled to photosynthesis [23] and to the tricarboxylic acid (TCA) 51
cycle in the mitochondria, providing the reducing power [24,25]. In addition, the TCA cycle 52
delivers energy and C skeletons for N uptake and assimilation [5,13,26]. N assimilation and 53
organic acid concentrations also follow a diurnal cycle. This has been demonstrated by higher 54
activities of NR and PEPC (i.e. the key enzymes in C and N physiology), higher 55
accumulation of malate, NH4+ and glutamine during the first part of the light period and in 56
contrast, a decrease in concentrations of citrate [26]. Given the different effects of both N 57
species on leaf gas exchange, metabolites and enzyme activities, it is likely to expect strong 58
and different effects of these N species on the C isotopic composition of plant material and 59
respired CO2. 60
Within this biochemical framework, the TCA cycle intermediate malate plays a central role.
61
Malate provides reducing equivalents for NO3- reduction as well as a redox storage in the 62
vacuoles [27,28]. It has also been shown that the TCA cycle is not fully functional during the 63
light period and consists of two loosely connected branches producing citrate and malate 64
[8,28–30]. According to Igamberdiev and Bykova [31], during the light period malate has the 65
potential to solely operate the TCA cycle by generating oxaloacetate (OAA) and pyruvate 66
(supplying both substrates of the TCA cycle) via malate dehydrogenase (MDH) and malic 67
enzyme (ME), respectively. Malate is also a key component in replenishing the loss of C 68
skeletons from the TCA cycle during the amino acid metabolism. This replenishment 69
happens through an anaplerotic reaction via phosphoenolpyruvate carboxylase (PEPC) [4]. In 70
addition, malate maintains the charge balance [32] and is involved in the re-transport of K+ 71
ions released after NO3- assimilation from shoots to the roots [21]. Together with citrate, 72
malate regulates the transfer of reducing power between cell compartments. Thus, both, 73
citrate and malate are expected to be central to unravel the underlying mechanisms if NO3-
74
vs. NH4+ supply affects the δ13C of foliage, metabolites and respired CO2 differently.
75
δ13C values in C3 plants depend primarily on the δ13C values of atmospheric CO2 and 76
photosynthetic isotope fractionation during CO2 assimilation mainly by RuBisCO [33].
77
Physiological responses such as variation in assimilation rates, mesophyll and stomatal 78
conductance can lead to changes in the ratio of leaf intercellular (Ci or more precisely 79
chloroplast CO2 concentration (Cc)) to atmospheric (Ca) CO2 concentrations (Ci/Ca), which in 80
turn changes δ13C of plant organic matter and subsequently that of respired CO2. 81
Consequently, δ13C values of bulk plant material, metabolites and respired CO2 vary under 82
environmental conditions, which alter the Ci/Ca ratio such as drought and/or temperature [34–
83
38], change over time and differ among species [39–47]. Additional “post-photosynthetic 84
isotope fractionations” caused by reactions during plant metabolism (e.g. during dark 85
respiration) also affect the δ13C of plant material [40,48,49]. Particularly, the anaplerotic 86
reaction of the TCA cycle, moderated via PEPC, results in a 13C enrichment of the introduced 87
COOH group (C-4) of OAA which often is converted to the more abundant metabolite like 88
malate [50,51] and eventually δ13C of respired CO2 [38,52]. However, although several 89
studies showed the influence of N source and content on C isotope discrimination during 90
photosynthesis [53–55], to date, there is no study explicitly investigating the effects of N 91
species supplied (changing from NO3- to NH4+) on the δ13C of plant metabolites and of 92
respired CO2. 93
Therefore, the overall aim of this study was to examine the effects of NO3- vs. NH4+ supply 94
on C isotope fractionation of leaf metabolites and dark respired CO2. We hypothesized a 95
change in δ13C of plant metabolites as well as respired CO2 in response to changing ratios of 96
NO3- and NH4+. Moreover, we hypothesized that malate and the anaplerotic reaction of the 97
TCA cycle play a central role for the fractionation during respiration of NO3- supplied plants.
98
To test our hypotheses, a series of climate chamber based experiments have been conducted 99
and relevant foliar gas exchange variables, enzymatic reactions, as well as concentrations and 100
δ13C values of selected metabolites and respired CO2 of Nicotiana sylvestris leaves have been 101
measured. Plants grew under a gradient of NO3- to NH4+ concentration ratios.
102
Materials and methods 103
Plant material 104
Tobacco seeds (Nicotiana sylvestris) were stratified for three days in water prior to sowing.
105
The seeds were then sown in organic substrate (substrate 2, Klassmann Deilmann, Germany) 106
and grown for two weeks in a walk-in climate chamber with average day/night temperatures 107
of 22/20 °C and relative humidity (RH) of 50-70%. Plants were exposed to 12 hours daylight, 108
supplemented by fluorescent lamps (Sylvania, Switzerland), with an average photosynthetic 109
photon flux density of approximately 400 µmol m-2 s-1 at the leaf surface. After two weeks, 110
each plant was moved to a 750 mL pot filled with sterile sand (Quarzsand A 0.7-1.2 mm, 111
Carlo Bernasconi AG, Switzerland), and perlite (Foma Sa, Switzerland) at the ratio of 5:1.
112
Five plants were used per treatment (see below), grown in individual pots.
113
Nutrients were supplied to plants with nutrient solutions modified according to Arnozis et al.
114
[56] as follows: 1 mM CaCl2; 1 mM MgSO4; 0.25 mM KH2PO4. Microelements and Fe- 115
EDTA were provided with identical concentrations as in a Hoagland solution. In order to 116
study the effect of different N species, 6 mM N was supplied to plants in different ratios of 117
NO3- to NH4+, provided in the forms of Ca(NO3)2, KNO3, (NH4)2SO4, and NH4NO3, while 118
keeping the total amount of N in the solution constant. In the 100% NH4+ nutrient solution an 119
additional 1 mM K2SO4 was added. Throughout the manuscript, when using the term ‘N 120
species gradient’, we refer to the different ratios of the two inorganic N species supplied.
121
Along this gradient, the following ratios were used: [%NO3--N: %NH4+-N] 100:0 (NO3-), 122
90:10 (90%), 75:25 (75%), 50:50 (50%), 25:75 (25%), 10:90 (10%), 0:100 (NH4+) shown in 123
Table 1. For simplicity, we will use the term “NO3- supply” when referring to plants fed with 124
100% NO3-, 90% NO3-, 75% NO3- and 50% NO3-, and will use the term “NH4+ supply” when 125
referring to plants fed with 75% NH4+, 90% NH4+ and 100% NH4+; sole NO3- and sole NH4+
126
treatments represent 100% NO3- and 100% NH4+ supply, respectively. Plants were treated 127
with the NO3-/NH4+ solutions (pH≈7) every second day. On the days without treatment, water 128
was provided for unlimited water supply. The pH of the drained soil solution was monitored 129
throughout the study. At harvest time, the pH value was ca. 6.5 in NO3- supplied plants and 130
ca. 5 in NH4+ supplied plants.
131
This setup was repeated three times and all plants were grown in identical conditions from 132
seeding, to ensure sufficient leaf material for prospective analyses. One set of plants was used 133
for leaf gas exchange measurements three hours after start of light period. The same leaves 134
were then used for 13C analysis of dark-respired CO2 and for the analysis of δ13C and 135
concentrations in organic matter and metabolites. The second and third sets were also 136
harvested three hours after the start of light period and used for measurements of respiration 137
and enzymatic analyses, respectively. All measurements and final harvest were performed 138
after 45 days of treatment (thus plants were about 8 weeks old at harvest time).
139
Foliar gas exchange variables 140
Net assimilation rate (An), ratio of intercellular CO2 to atmospheric CO2 concentration (Ci/Ca) 141
and stomatal conductance (gs) were measured with a LI-6400 (LI-COR, USA) on one mature, 142
fully expanded leaf attached to the plant. A standard photosynthesis cuvette with a leaf area of 143
6 cm2 (6400-02B LED Light Source) was used while leaf temperature was set to 20 °C. PAR 144
(Photosynthetically Active Radiation) was set at 400 µmol m-2 s-1 and [CO2] (Ca) was kept at 145
400 µmol mol-1 with a constant flow rate of 500 µmol s-1. After the plants were in steady state 146
(An and gs stabilized), each gas exchange measurement took 60 seconds with the data 147
recorded every 10 seconds and averaged afterwards. The remaining plant leaves were shock- 148
frozen in liquid N2. The dry weight of the leaves was measured after freeze drying to 149
determine plant foliar biomass. These samples were then ground to a fine powder using a ball 150
mill (MM 200, Retsch, Germany) and stored in a desiccator for further analyses.
151
δ13C values of leaf respired CO2 and respiration rates 152
The C isotopic composition of leaf dark-respired CO2 was measured during the day similar to 153
previous studies [52,57,58]. After the measurement of gas exchange, the respective leaf was 154
cut and immediately transferred into a gas tight Tedlar® bag (Keyka Ventures, USA) 155
according to Barbour et al. [59] and Barthel et al. [60]. To avoid the light enhanced dark 156
respiration (LEDR) period, the Tedlar bag containing the leaf was kept in the dark for 30 min 157
before measurements started [36,61–63]. The sampling bag was flushed several times with 158
synthetic CO2-free air (20% O2 and 80% N2, Pangas, Switzerland) until a CO2 free 159
atmosphere was established, controlled by an infrared gas analyser (LI-820, LI-COR, USA).
160
The bag with the leaf was kept in the dark for approximately one hour and the air was 161
sampled with a gas-tight syringe (BD Plastipak, Switzerland) and transferred into a 12 mL 162
evacuated gas-tight glass vial (“Exetainer”, Labco, England). The δ13C value of dark respired 163
CO2 (δ13CR) was measured with a modified Gasbench II as described by Zeeman et al. [64]
164
coupled to a DeltaplusXP isotope ratio mass spectrometer (IRMS, ThermoFisher, Germany). In 165
addition, the δ13C value of atmospheric CO2 in the climate chamber was determined as -10.6 166
mUr ± 0.9 (SD). All C isotope measurements in CO2 had a typical standard deviation of less 167
than 0.15 mUr.
168
For measurements of respiration rates, plants were also kept in the dark for 30 mins to avoid 169
LEDR. A fully developed leaf was cut and put into a 445 mL glass jar covered with an opaque 170
plastic foil. The lid of the jar was equipped with a luer-stopcock fitting to allow gas sampling.
171
During a one-hour period after closing the jar, 4 samples were taken. The first sample was 172
taken right after closing the jar, and the following three samples were taken every 20 minutes.
173
CO2 concentrations were measured with a gas chromatograph (456-GC, Scion Instruments, 174
UK). After CO2 sampling, the leaf was scanned (Lide 20, Canoscan, Canon) and the leaf area 175
determined with the software package Fiji [65]. Leaf respiration rates were derived from the 176
linear increase in CO2 concentrations over time. Respiration rates accounted for leaf area, 177
head space volume and temperature (ideal gas law; cf. [66]).
178
δ13C values of bulk organic matter 179
After freeze-drying and milling, approximately 2 mg of bulk leaf material was weighed in Sn 180
capsules (5 x 9 mm, Saentis, CH) for δ13C analysis of bulk leaf organic material (δ13CB) with 181
a Flash EA 1112 Series elemental analyzer (ThermoFisher, Germany) coupled to a 182
DeltaplusXP IRMS via a ConFlo III as described by Brooks et al. [67] and Werner et al. [68].
183
Sequencing and referencing followed the recommendation by Werner and Brand [69]. The 184
measurement precision (SD) of the quality control standard (tyrosine Tyr-Z1) during the δ13C 185
measurements of all EA samples reported in this study was 0.16 mUr.
186
Organic metabolites for bulk δ13C analysis 187
Extraction of organic metabolites was performed according to Lehmann et al. [38]. Briefly, 188
100 mg freeze-dried plant material were transferred to 2 mL reaction tubes. 1.5 mL 85 °C 189
deionized water was added to prevent any enzymatic activity. The samples were then 190
incubated for 30 min in a water bath at 85 °C. After centrifugation for 2 min at 10000 g, the 191
supernatant and the remainder (pellet) were separated and kept for further purifications. Ion 192
exchange chromatography was used to separate the supernatant containing the water-soluble 193
content into sugars, amino acids, and organic acids fractions. 1 mL of the supernatant was 194
added to a column filled with a cation exchanger (Dowex 50WX8, hydrogen form, 100-200 195
mesh, Sigma Aldrich, CH), that was directly above a second column filled with an anion 196
exchanger (Dowex 1X8, chloride form, 100-200 mesh, Sigma Aldrich, CH). The neutral sugar 197
fraction was eluted with 30 mL deionized water. The amino acid fraction was trapped on the 198
Dowex 50WX8 and then eluted with 30 mL 3 M ammonia solution, while the organic acid 199
fraction was trapped on the Dowex 1X8 column and afterwards eluted with 35 mL 1 M HCl 200
[38,70]. All metabolic fractions were frozen, freeze dried, re-dissolved in deionized water and 201
stored at -20 °C for further use.
202
Starch was enzymatically isolated from the pellet originating after the water extraction 203
mentioned above, according to standard protocols [70,71]. In brief, the pellet was washed 204
several times using an MCW solution (methanol/chloroform/deionized water, 12/5/3, v/v/v) 205
and deionized water, and subsequently dried overnight. On the second day, the starch in the 206
pellet was re-solubilized in water and gelatinized at 100 °C for 15 min and broken down to 207
soluble sugars at 85 °C for 2 hours using the heat resistant -amylase (EC 3.2.1.1, Sigma- 208
Aldrich, CH). The -amylase was separated from the soluble sugars by centrifugation filters 209
(Vivaspin 500, Sartorius, Germany). For the respective δ13C measurements, aliquots from the 210
starch-derived sugar (δ13CSt), sugar (δ13CS), amino acid (δ13Caa), and organic acid (δ13Coa) 211
fractions were pipetted into Sn capsules, dried in an oven at 60 °C, and analysed with the EA- 212
IRMS coupling as described above. Compound specific isotope analysis (CSIA) of individual 213
organic acids was performed following Lehmann et al. [38] and Rinne et al. [72] and CSIA of 214
individual amino acids was performed following Schierbeek [73] and Vlaardingerbroek et al.
215
[74]. Detailed descriptions of these methods are provided in the supplementary information 216
section.
217
Enzymatic activities 218
The leaf material for enzyme activity assays was frozen and immediately ground with pestle 219
and mortar in liquid nitrogen, then stored at -80 C until analyses. The activities of NR, PEPC 220
and ME were assayed following Dier et al. [75], Gibon et al. [76] and Tronconi et al. [77], 221
respectively. Detailed descriptions of these methods are provided in the supplementary 222
information section.
223
Concentration of carbohydrates 224
Glucose, fructose, sucrose, total sugars and total starch were extracted from the leaves 225
according to Ruckle et al. [78]. In brief, 10 mg of freeze-dried leaf material was washed two 226
times with 80% (v/v) ethanol and two times with 50% (v/v) ethanol to separate the soluble 227
sugars from starch. Each ethanol extract was incubated at 80 °C for 30 min. The remaining 228
pellet containing starch was incubated at 90 °C in 0.2 M KOH and then neutralized with 1 M 229
acetic acid. The starch in the pellet was digested with -amylase and amyloglucosidase 230
(Sigma Aldrich, CH) to convert it to glucose. Starch-derived glucose, glucose, fructose and 231
sucrose were quantified in triplicates using an enzyme-linked assay based on hexokinase, 232
glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, and invertase as described by 233
Ruckle et al. [78]. Sugar dependent conversion of NADP+ to NADPH (Sigma Aldrich, CH) 234
was quantified using a plate reader (Perkin Elmer Enspire©, USA) by measuring the 235
absorption at 340 nm.
236
Statistics and calculations 237
To study the magnitude of post-photosynthetic C isotope fractionation we calculated the 238
apparent respiratory isotope fractionation according to Ghashghaie et al. [79]:
239
ℯdark= (𝛿13CS - 𝛿13CR) / (1 + 𝛿13CR) 240
where δ13CS andδ13CR represent the isotopic composition of soluble sugars and the isotopic 241
composition of respired CO2, respectively. We used the simplified Farquhar model 242
= a + (b-a) * Ci/Ca
243
Where a is the isotope fractionation during diffusion and b during carboxylation to calculate 244
carbon isotope discrimination [34]. We tested the effect of different N species solutions on 245
enzyme activities, concentrations and δ13C values with one-way ANOVA using R [80]. The 246
different solutions (NO3- to NH4+) were treated as factors. Tukey-HSD post-hoc tests were 247
used for multiple comparisons at the 0.05 significance level. We used linear regression 248
models to calculate the p and R2 values given in the graphs; asterisks represent different 249
significance levels: * for p<0.05, ** for p<0.01 and *** for p<0.001. All δ13C values were 250
measured on V-PDB scale.
251
Results 252
Foliar gas exchange variables 253
The change in N species supplied led to a shift in gas-exchange behaviour in tobacco plants 254
(Tab. 2). We found a significant effect of the N species gradient on net assimilation rate (An), 255
with approximately 50% lower An under NH4+ compared to NO3- supply (Fig. 1A, p=0.012).
256
The ratio of intercellular to atmospheric CO2 concentrations (Ci/Ca) was higher with 257
increasing NH4+ fraction (Fig. 1B, p=0.0014). Although stomatal conductance (gs) was more 258
variable under NH4+ than under NO3- supply, no significant treatment effect was observed 259
(Fig. 1C, p=0.6). In contrast, respiration rate (Rdark) significantly increased when NH4+ supply 260
increased (Fig. 1D, p<0.01), whereas leaf dry biomass significantly decreased with increasing 261
NH4+ (Fig. 1E, p<0.001).
262 263
Carbon isotopic composition of plant metabolites and respired CO2 264
C isotopic composition of leaf bulk organic matter (δ13CB) stayed relatively constant at about 265
-27.4 mUr from 90% NO3- to 10% NO3-, but tended to be lower in 100% NO3- and 100%
266
NH4+ with -28.3 mUr and -29.1 mUr, respectively (Tab. 2, Fig. 2A). Moreover, we did not 267
find a relationship between δ13CB and N content (data not shown). δ13C of leaf metabolites 268
(e.g. sugars, starch and organic acids) and leaf respired CO2 were clearly affected by changes 269
in the N species ratios (Fig. 2). δ13C values of sugars (δ13CS) increased steadily from -27.3 270
mUr (100% NO3-) to -25.8 mUr (10% NO3-) but decreased sharply to -28.4 mUr under 100%
271
NH4+ (Fig. 2B). Also, δ13C of starch (δ13Cst) increased from -27.2 mUr (100% NO3-) to -25.9 272
mUr (50% NO3-) before decreasing to -27.8 mUr (100% NH4+). No significant differences 273
were observed for isotopic composition of amino acids (δ13Caa: Fig. 2D, p=0.14). δ13C values 274
of organic acids (δ13Coa) and respired CO2 (δ13CR) followed similar patterns along the N 275
species gradient from NO3- to NH4+, with almost steadily decreasing δ13C values, which were 276
also less negative compared to those of other metabolic fractions particularly under NO3-
277
supply (Figs. 2E,F). Both δ13Coa and δ13CR showed more negative values under NH4+ supply 278
compared to those under NO3- supply, ranging from -24.2 mUr (δ13Coa in 90% NO3-) to -27.8 279
mUr (δ13Coa in 100% NH4+), and from -24.4 mUr (δ13CR in 90% NO3-) to -27.8 mUr (δ13CR in 280
100% NH4+), respectively.
281 282
Compound-specific analysis of organic acids 283
Malate and citrate were the dominant organic acids in the bulk organic acids extracted for 284
compound specific analysis. δ13C values of malate tended to be more negative under NO3-
285
than under NH4+ supply, gradually increasing from -21.6 mUr to -19 mUr, before decreasing 286
again at 100% NH4+ (Tab. 2, Fig. 3A, p=0.07). In contrast, δ13C values of citrate showed a 287
clear effect of N species, with δ13C values decreasing from -22.9 mUr in 90% NO3- to -26.9 288
mUr in 10% NO3- treatment (Fig. 3B, p=0.017). Moreover, very strong effects were observed 289
in malate concentrations, which decreased with increasing NH4+ from 128 µmol g DW-1 in 290
100% NO3- to about 2 µmol g DW-1 in 25% NO3- (Fig. 3G, p<0.001). Similarly, citrate 291
concentrations decreased from 47 µmol g-1 in 75% NO3- to less than 1 µmol g DW-1 in 10%
292
NO3- (Fig. 3H, p<0.001). It has to be noted that concentrations and δ13C values need to be 293
treated with caution since the concentrations of malate and citrate were at the detection limit 294
of HPLC-IRMS measurements for plants supplied with less than 50% NO3- and should be 295
interpreted with caution.
296 297
Compound specific analysis (CSIA) of amino acids 298
In this study, we focused on four amino acids including alanine (related to pyruvate), serine 299
(a product of photorespiration in leaves), asparagine/aspartic acid (related to OAA) and 300
glutamine/glutamic acid (related to 2-oxoglutarate). N species treatments significantly 301
influenced δ13C values of alanine, asparagine and serine (Fig. 3C-E, p<0.001 for alanine and 302
serine, p=0.005 for asparagine), but not those of glutamine (Fig. 3F). While δ13C of alanine 303
was gradually decreasing with increasing NH4+ supply, with highest values in 100% NO3- (- 304
27.9 mUr) and lowest in 100% NH4+ (-29.5 mUr, Fig. 3C), no clear pattern was observed 305
along the N species gradient for asparagine (Fig. 3D). Similar to alanine, serine was more 306
enriched in 13C in plants grown under NO3- supply, with highest δ13C value in 90% NO3- (- 307
27.8 mUr), and more depleted in 13C under NH4+ supply, with the lowest δ13C value in 10%
308
NO3- (-32.5 mUr, Fig. 3E). Furthermore, concentrations of all four amino acids increased 309
from NO3- supply towards 10% NO3-, before sharply decreasing again at 100% NH4+, almost 310
reaching the same values as under NO3- supply (Figs. 3 I-L, p<0.01). The N species effect 311
was very clear in alanine and also in serine, with the highest concentrations of alanine in 10%
312
NO3- (2.8 µmol g DW-1) and lowest in 100% NO3- (0.5 µmol g DW-1) and highest 313
concentrations of serine in 10% NO3- (23.7 µmol g DW-1) and lowest in 90% NO3- (2.6 µmol 314
g DW-1). The influence of N species on the concentrations of asparagine and glutamine was 315
not as clear, although NH4+ supplied plants showed higher concentrations compared to NO3-
316
supplied plants.
317 318
Enzyme activity 319
Enzymatic activities of NR, PEPC and ME were negatively affected by the gradient of NO3-
320
to NH4+ supply (Fig. 4, p<0.001), most clearly seen in NR and PEPC. As the fraction of NH4+
321
increased, NR activity decreased from 1.9 nmol NO2- g FW-1 s-1 (100% NO3-) to 0.7 nmol 322
NO2- g FW-1 s-1 (10% NO3-, Fig. 4A). Similarly, PEPC activity decreased from 9.5 nmol 323
NAD g FW-1 s-1 (100% NO3-) to 4.3 nmol NAD g FW-1 s-1 (100% NH4+, Fig. 4B). In contrast, 324
ME activity was lowest under 25% NO3- (269 nmol NAD g FW-1 s-1) and highest under 50%
325
NO3- supply (505 nmol NAD g FW-1 s-1, Fig. 4C). The activities of NR and ME in plants 326
growing under 100% NH4+ could not be analysed due to insufficient amount of plant 327
material.
328 329
Concentration of carbohydrates 330
The gradient of N species from NO3- to NH4+, strongly influenced concentrations of both 331
water-soluble carbohydrates and starch (Fig. 5). Concentrations of glucose, fructose, sucrose 332
and total sugars significantly increased as the percentage of NH4+ in the N supply increased, 333
except under 100% NH4+ (Fig. 5A-D). In contrast, starch concentrations were highest in 334
plants supplied with higher percentage of NO3-, with concentrations decreasing from 359 mg 335
g DW-1 in 50% NO3- to 95 mg g DW-1 under 10% NO3- supply (Fig. 5E).
336 337
Potential substrates for respiration and apparent isotope fractionation 338
As stated above, leaf respiration rates as well as δ13CR were influenced by the gradient of N 339
species. In addition, we found a significant and positive relationship between δ13CR and 340
δ13Coa, with a slope close to one and an intercept close to zero, representing a 1:1 relationship 341
between these two variables (Fig. 6, R2=0.93, p<0.001, y = 0.97x - 0.72). This relationship 342
clearly showed that both organic acids and respired CO2 became more depleted in 13C along 343
the N species gradient from NO3- to NH4+. 344
Also, apparent isotope fractionation (ℯdark), calculated from δ13CS and δ13CR, was influenced 345
by the N species gradient, with negative ℯdark values indicating higher 13C enrichment in 346
respired CO2 compared to sugars as the first products of photosynthesis. ℯdark in plants 347
supplied with more than 50% NO3- was more negative compared to plants supplied 348
dominantly with NH4+ (Fig. 7). ℯdark values were the lowest (-2.4 mUr) in plants of the 90%
349
NO3- treatment and increased along the gradient to the highest values in plants under 10%
350
NO3- (0.9 mUr).
351
Discussion 352
NO3- to NH4+ gradient affects foliar gas exchange and metabolism 353
Our main aim was to study C isotope fractionation in response to a supply gradient of two 354
different N species. We therefore setup an experiment with tobacco plants supplied with a N 355
gradient from 100% NO3- to 100% NH4+. As expected, foliar gas exchange characteristics 356
and biomass were strongly influenced by this N supply gradient (Fig. 1). As NO3- decreased, 357
net assimilation rate also decreased in our tobacco plants (Fig. 1A), similar to earlier studies 358
with spinach [81], sugar beet [82] and maize [83]. The decreasing net assimilation rate and 359
the simultaneously increasing respiration rate along the gradient (Fig. 1D), can explain the 360
observed lower biomass in plants under NH4+ supply (Fig. 1E), most likely a sign of NH4+
361
toxicity caused by NH4+ supply [16, 12, 18], supported by the lower pH values (by 1.5) we 362
observed in the drained soil solution from NH4+ supplied plants. Moreover, lower 363
assimilation rate with decrease in NO3- supply is in line with the lower needs for reducing 364
equivalents.
365
We observed strong impacts of the ratio of N species supplied on all measured metabolite 366
concentrations such as organic acids (malate and citrate), soluble sugars, starch and amino 367
acids (alanine, serine, asparagine and glutamine), as well as on measured enzyme activities.
368
As suggested by the higher assimilation of plants dominantly supplied with NO3-, there was 369
probably a higher supply of reducing power, in line with the observed higher NR activity 370
(Fig. 4A). Decreasing PEPC and ME activities (Fig. 4B,C) together with decreasing 371
concentrations of organic acids along the gradient from NO3- to NH4+ (Fig. 3G,H) imply an 372
important role of organic acids (e.g. malate) and the anaplerotic reaction of the TCA cycle 373
under NO3-, but not under NH4+ supply [5,29,55]. Altogether, these findings clearly 374
confirmed that a change in N species supplied to plants has an influence on carbon 375
metabolism and thus on leaf respiratory metabolism.
376
We further observed higher concentrations of soluble sugars in the leaves of plants supplied 377
dominantly with NH4+ (Fig. 5D) which aligned with higher respiration rates in these plants 378
(Fig. 1D), resulting in a strong positive relationship between sugar concentrations and 379
respiration (R2=0.75, p<0.05, Fig. S1). This might be a stress response to NH4+ toxicity, 380
supported by suppressed growth observed in this study as also suggested by Hachiya et al.
381
[84]. N assimilation under high NH4+ supply takes place mostly in the roots [17,19] and 382
requires C skeletons to be delivered from the leaves [85]. However, not only aboveground 383
but also root biomass was low in NH4+ supplied plants (data is not shown) [86].
384
Moreover, the N species gradient not only influenced concentrations of organic acids and 385
carbohydrates, but also the concentrations of individual amino acids (Fig. 3). We observed an 386
increase in concentrations of alanine, serine, asparagine and glutamine with increasing NH4+
387
in the supply solutions (Fig. 3 I-L), supporting previous findings [2,22,87]. These 388
observations might simply be signs of a detoxification strategy, i.e., remove free NH4+ ions 389
from the cytosol by converting them into amino acids [19,22,88]. Moreover, decreasing C:N 390
ratios are reported to relate to increasing amino acid biosynthesis [24,87], as seen for the 391
NH4+ supplied plants in our study (Fig. S2 A). Consequently, we are confident that our 392
experimental setup allowed in-depth assessment of the C isotopic composition of metabolites 393
and respired CO2 in relation to a N species gradient.
394 395
NO3- to NH4+ gradient influences δ13C of amino acids 396
Moreover, we conducted compound specific isotope analysis of individual amino acids to 397
investigate if changes observed in the post-photosynthetic fractionation between NO3- and 398
NH4+ supplied plants were mirrored in the δ13C of individual amino acids as the products of 399
N assimilation. Analysis of amino acids showed different effects of the two N species on δ13C 400
values of individual amino acids, particularly of alanine and serine (Fig. 3C,E; except for 401
serine under 100% NH4+ supply). A possible explanation for 13C enriched alanine in NO3-
402
supplied plants can be the isotope fractionation caused by pyruvate dehydrogenase (PDH), 403
resulting in an accumulation of 13C enriched glycolytic pyruvate that is known as a precursor 404
for alanine biosynthesis [89,90]. Alternatively, the observed 13C enriched alanine in NO3-
405
supplied plants can be explained by higher ME activity (Fig. 4C), supplying amino acid 406
biosynthesis with 13C enriched pyruvate from anaplerotically synthesized malate.
407
δ13C values of serine and alanine were similar in NO3- supplied plants (around -27 mUr), but 408
serine was by 3 mUr more 13C depleted in NH4+ plants compared to alanine (-32.5 mUr to - 409
29.5 mUr, respectively). Tcherkez [91] suggested a 13C depletion in serine molecules 410
compared to glycine in relation to photorespiration. Our findings therefore can convey 411
possible higher photorespiration in NH4+ supplied plants, resulting in 13C depletion of serine.
412
Although δ13C of asparagine was not influenced by the gradient of N species, it showed the 413
highest 13C enrichment among all other amino acids. Sechley et al. [92] underlined the role of 414
OAA/malate as donors of C skeleton for synthesis of asparagine. Given that malate was the 415
most 13C enriched compound in our study (Fig. 3A), a contribution of this compound to the 416
13C enrichment of asparagine is likely.
417 418
NO3- to NH4+ gradient influences carbon isotope fractionation 419
We observed distinct changes in δ13C values of respired CO2 and various metabolites such as 420
soluble carbohydrates, starch, organic acids and consequently leaf bulk organic matter in 421
relation to the N species gradient (Fig. 2). Despite the clear increase of Ci/Ca ratio from NO3-
422
to NH4+ supplied plants (Fig. 1B), suggesting larger 13C discrimination and therefore more 423
13C depleted assimilated along the gradient, the δ13C values of sugars, starch and bulk organic 424
matter showed no significant response (Fig. 2A,B). Therefore, we could not explain the 425
differences between δ13C values of metabolites (sugars and starch as the first products of 426
photosynthesis) along the gradient (R2<0.54) using the Ci/Ca ratio alone, based on the 427
simplified Farquhar model (= a + (b-a) * Ci/Ca, Fig. S3, [34]). This might have several 428
reasons: (1) N concentration positively influences mesophyll conductance and thus Cc/Ca, 429
resulting in higher 13C discrimination which in turn causes more 13C depleted sugars 430
[43,45,93,94]. However, although we found higher N concentration in the leaves of NH4+
431
supplied plants (as shown in Fig. S2 B) and thus potentially increased mesophyll 432
conductance, soluble sugars in NH4+ supplied plants were not depleted in 13C. (2) The foliar 433
sugar pool has accumulated over longer time periods than can be represented by individual 434
snap-shot Ci/Ca measurements. (3) According to Caemmerer and Evans [43], there is a 435
possible influence of PEPC activity on the expected C isotope discrimination during 436
carboxylation by RuBisCo. Considering the different PEPC activities measured in NO3- and 437
NH4+ supplied plants, such an effect of PEPC on discrimination cannot be excluded.
438
To further assess the mechanisms underlying the effects of NO3- vs. NH4+ supply on the 439
fractionation during respiration, we calculated the apparent isotope fractionation (ℯdark) 440
between the sugars δ13CS (as the first photosynthetic product) and respired CO2 (δ13CR) 441
according to Ghashghaie et al. [79]. Interestingly, ℯdark values were much more negative in 442
plants supplied with more than 50% NO3- than in plants supplied predominantly with NH4+
443
(Fig. 7). This implies that in plants supplied with NO3-, substrates for the respiration were 444
more enriched in 13C relative to sugars (as the first products of photosynthesis and main 445
metabolites in the respiratory pathway) which in turn resulted in the observed 13C enrichment 446
of respired CO2. In contrast, plants with higher percentage of NH4+ supply showed ℯdark
447
values which were on average close to or slightly above zero, reflecting a very minor and 448
thus negligible difference between δ13CS and δ13CR (Fig. 7). These results confirmed our 449
hypothesis that a shift from NO3- to NH4+ supply mainly influences post-photosynthetic 450
isotope fractionation.
451
The high concentrations of malate and the high PEPC activity in NO3- supplied plants 452
together with relatively high δ13C values of malate amongst all other metabolites (Fig. 3A) 453
further support the idea that the anaplerotic reaction of the TCA cycle could play an 454
important role in the 13C enrichment of CO2 respired by the leaves of NO3- supplied plants. In 455
order to determine the relative contribution of the anaplerotic reaction responsible for the 13C 456
enrichment in leaf-respired CO2, we calculated the possible 13C enrichment of the C-4 atom 457
in malate [95]. According to the model proposed by Mezler and O’Leary [95], the carbon 458
introduced into malate via PEPC is about 6 mUr more enriched in 13C than that of 459
intercellular (internal) CO2. After calculating the δ13C of intercellular CO2, we adjusted the 460
Melzer and O’Leary model by assuming that in plants supplied with 100% NO3-, PEPC has 461
the highest activity (supported by our own results, Fig. 4) and therefore these 100% NO3-
462
plants have the highest enrichment of the C-4 atom in malate (i.e. an additional 6 mUr). The 463
13C enrichment of all other N treatments along the gradient were then calculated by using the 464
ratio of their PEPC activity compared to that of 100% NO3- supplied plants (Fig. S4). Our 465
results showed a clear decrease in the 13C enrichment of the C-4 atom in malate along the N 466
species gradient. These findings indicated that a higher proportion of anaplerotically 467
introduced carboxyl group(s) in malate might be released as CO2 during amino acid 468
biosynthesis or plant dark respiration, particularly in NO3- supplied plants (Fig. 8). We 469
therefore conclude that such a change in the PEPC related mechanism along the N species 470
gradient is the most likely pathway to explain 13C enrichments observed in δ13C of organic 471
acids and of respired CO2 under NO3- supply (see Fig. 6).
472 473
Conclusion 474
This study aimed to understand the influence of a supply gradient in NO3- and NH4+ supply 475
on the C isotope fractionation as seen in leaf metabolites and during leaf respiration. Several 476
lines of evidence suggest that modifications of the N species supplied to plants, which 477
changed foliar gas exchange, enzymatic activities and concentrations of metabolites (i.e.
478
sugars, amino acids and organic acids), also changed the C isotopic composition of leaf 479
metabolites and respired CO2, supporting our first hypothesis. With our second hypothesis, 480
we suggested a major role of the anaplerotic reaction of the TCA cycle (via PEPC) and 481
organic acids. Indeed, our results indicated a higher contribution of anaplerotically 482
assimilated carbon in the process of dark respiration in plants supplied dominantly with NO3-, 483
leading to enrichments in metabolites and respiratory CO2. Our findings on the impact of N 484
species on δ13C of organic acids and respired CO2 might gain additional relevance since the 485
N species available to vegetation is strongly affected by either management or anthropogenic 486
pollution while δ13C values of leaf metabolites or respiration are widely used in plant 487
sciences, e.g. as a proxies for plant physiological responses to environmental change or to 488
partition ecosystem CO2 fluxes.
489 490
Acknowledgments 491
The authors thank A. H. Khoshgoftarmanesh from Isfahan University of Technology for 492
helping with the calculation of the salts needed for the gradient of nutrient solution. We also 493
acknowledge the help of A. Ackermann for EA-IRMS measurements in the isotope 494
laboratory of the Grassland Sciences Group at ETH Zurich. We appreciate statistical help 495
given by C. Bachofen from the Grassland Sciences Group. We would like to thank B. Studer 496
for use of laboratory space, reagents, and equipment for the carbohydrate analysis from the 497
Molecular Plant Breeding group at ETH Zurich. We would like to thank H.J Lendi (ETH 498
Zurich) for technical support of the climate chambers and freeze-dryers.
499 500
Declaration of authorship 501
SG, RAW, NB, FWB, JG, MML, RTWS developed the presented idea, planned, and designed the 502
research. SG conducted the experiments as well as data analysis and wrote the manuscript. SG, RH, 503
RM, SS, MH performed enzyme activity assays and MER and DAC contributed in carbohydrate 504
concentration measurements. MML carried out compound specific analysis of organic acids. All 505
authors discussed the results and contributed to the final manuscript.
506 507
Funding 508
SG was funded by the Swiss National Science Foundation (project 205321_153545 “CarIN”) granted 509
to RAW and RTWS. This research was partially supported by the COOP—Research Fellowship 510
Program (HERC) through the ETH-World Food System Center and the Swiss Federal Office for 511
Agriculture (FOAG) granted to MER and by the SNF Ambizione project “TreeCarbo”
512
(PZ00P2_179978) granted to MML.
513 514
Supplemental Material 515
The following supplemental materials are available.
516
Figure S1. Linear regression between respiration rate (µmol m-2 s-1) and concentration of 517
soluble sugars (mg g DW-1) 518
Figure S2. Carbon to nitrogen ratio (C:N) and nitrogen concentration (%) 519
Figure S3. Linear regression between measured 𝛿13C of starch/sugar and modeled 𝛿13C 520
values using the simplified Farquhar model 521
Figure S4. Modelled carbon isotopic composition of C-4 of malate in the leaves of tobacco 522
plants 523
524
Methods of compound-specific δ13C and concentration analysis of amino acids and organic 525
acids 526
Enzyme activity analysis: NR, PEPC and ME 527
528
Conflict of Interest Statement 529
The authors declare that the research was conducted in the absence of any commercial or 530
financial relationships that could be construed as a potential conflict of interest.
531
532
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