Introduction
Nitrogen (N) is a key
mineral for plant growth as well key factor limiting crop yield
(Liu et al. 2017). Under nitrogen deficiency,
plants will cause the lower leaves to chlorosis, limit the growth of buds and
reduce the growth of plants (Zhao et al. 2020;
Mu and Chen 2021). In the field, a large amount of nitrogen fertilizer
is applied to increase the yield. In the past decade, the
amount of N fertilizer applied in China has increased, with the application
amount accounting for more than 35% of the world’s total, making China the
largest N consumer in the world (Wu et al. 2017). However, crops have limited N fertilizer
absorption efficiencies, leaving large amounts of N fertilizers to pollute the atmosphere in the form of nitric oxide and
nitrogen dioxide. Additionally, the excess decreases water quality and pollutes
rivers as nitrate nitrogen and ammonia nitrogen (Wang et al. 2013). This not only causes environmental pollution,
but also increases the production costs of crops. Therefore, cultivating low
nitrogen (LN)-tolerant varieties, improving N use efficiency and reducing N use
are effective strategies for sustainable agriculture (Zhao et al. 2019).
Soybean (Glycine max L.) is an important food and
oil crop worldwide, providing 69% of the world’s dietary protein and 30% of the
edible oil. Wild soybean (W) is the ancestor of the cultivated soybean (M), it
has the benefits of high protein content, wide distribution, and strong stress
resistance (Kong
et al. 2017). Moreover, there
is no inter-specific hybridization barrier between them, allowing wild soybean
to play a key role in the genetic improvement and the selection of elite
varieties of cultivated soybean (Liu et al.
2019). Previous studies have shown that wild soybean has a stronger tolerance to LN stress than
cultivated soybean (Li et al. 2017).
Therefore, it is necessary to systematically study the mechanism of wild
soybean resistance to LN stress from the perspective of carbon (C) and N
metabolism.
The main plant organs for the
absorption, transformation and transmission of energy are the leaves, and their
functional characteristics to directly reflect plant hereditary characteristics
and the effective utilization of resources (Guo et al. 2016). The functional characteristics of the leaves vary
with location, which affects the exchange of substances and energy between the
plant and the surrounding environment (Wang et
al. 2012a), as well as the plant’s survival strategy, which is formed to
adapt to changes in the environment. The function,
structure and activity of young and old leaves are different, old leaves are
sacrificed to ensure the normal growth and development of young leaves (Shen et al. 2021). Previous studies have
found that under low nitrogen stress, nitrogen in old leaves can be transported
to new leaves for growth (Feller and Fischer 1994); chlorophyll binding protein
can be induced in new leaves, which is conducive to the recovery and reuse of
nitrogen in old leaves (Avice and Etienne 2014). Therefore, studying the
photosynthetic characteristics, C and N metabolism of wild soybean and
cultivated soybean under LN-stress conditions in young and old leaves can
provide new information to improve soybean production.
In this study, wild soybean and
cultivated soybean were selected as experimental materials and treated with an
artificial simulation of LN stress. Biomass, ion accumulation, photosynthetic
parameters and key N metabolism-related enzymatic activities were investigated
in wild soybean and cultivated soybean under LN stress. The main purposes of
this experiment were to study the physiological adaptation mechanisms,
differences between wild and cultivated soybean under LN stress. Additionally,
we investigated how to use wild soybean to improve cultivated soybean to
cultivate LN-tolerant crop varieties by measuring photosynthetic pigments and
its gas exchange traits, key enzymes of nitrogen metabolism, thereby revealing
the dynamic response processes of the C–N coupling relationship.
Materials and Methods
Plant materials
The seed of wild (‘Huinan06116’) and cultivated
soybean (‘Jinong24’) were provided by the Jilin Center of Germplasm
Introduction and Breeding of Crops, Changchun City, China.
Plant growth conditions and stress treatments
The wild and cultivated soybean seeds were
planted in a 14 cm diameter plastic pot containing 2.5 kg of washed sand and
germinated in water. All test materials are rainproof. After seedling
emergence, they were watered with 1×Hoagland’s solution every morning. The
plants were grown in the outdoor experimental fields, and the night and day
temperatures was 17.0–20.0°C and 24.0–28.0°C, respectively, and a relative
humidity of 55–65% at Northeast Normal University, Changchun City, Jilin
Province, China.
The LN treatment was initiated when the third compound leaf emerged.
Seeds of wild and cultivated soybean from the LN treatment group were grown in
the modified Hoagland solution of 1/2 intensity (N1), 1/3 intensity (N2) and
1/4 intensity (N3) for 2 weeks, respectively. CK culture under normal
conditions (1 × Hoagland solution). Wild and cultivated soybean were divided
into 4 groups: control (cK) and treatments N1, N2 and N3. Each group of 8 pots:
4 pots were used to measure growth and photosynthesis parameters, 4 pots were
used to analyze ion content and enzyme activity level (Table 1).
Measurement of growth, Total C and N contents
After two weeks of stress
treatment, soybean plants were harvested. The plant heights, root lengths,
above-ground fresh weights (Up FWs), below-ground FWs (Under FWs), above-ground
dry weights (Up DWs), and below-ground DWs (Under DWs) were measured (Shi et al. 2015). A
stable isotope mass analyzer (isprime Element Analyzer, isprime Ltd., Japan)
was used to evaluate the total nitrogen and carbon content (%) of the seedling
leaves through a 1 mg dry powder sample. SigmaPlot 10.0 (Systat Software Inc.)
and SPSS 16.0 (SPSS Inc.) software were used to analyze the experimental data
(Leticia et al. 2019).
Photosynthetic indices measurements
After two weeks of treatment,
the first two leaves at the top and the first two leaves at the bottom were
selected from the four pots to represent enough new and old leaves. The LI-6400
portable open gas exchange system (LI-COR, USA) was used to measure the
photosynthesis rate (PN), stoma conductivity (gs) and
leaf transpiration rate (E) at 11:00 in the morning. Atmospheric CO2
concentration, effective photosynthetic radiation, temperature and air humidity
were 375~385 cm3 m-1, 1150~1250 µmol m-2 s-1, 24°C and 50% (Li et al. 2018).
Dried leaf samples (30 mg) were
immersed in 10 mL of a mixture of 80% acetone: absolute ethanol (1: 1) and
photosynthetic pigments were extracted in the dark at room temperature until
the leaves turned white. Three spectrophotometric
measurements (Spectrov-754, Shanghai Accurate Scientific Instruments) were
performed on each sample at 440, 645 and 663 nm using Holm’s (1954) formula.
The content of photosynthetic pigments were calculated in mg g-1,
photosynthetic pigments included chlorophyll a (Chl a),
chlorophyll b (Chl b), chlorophyll a + chlorophyll b[Chl(a+b)]
and carotenoids (auto) (Jiao et al.
2018).
To 50-mg dry samples, 4 mL of deionized water
was added. Samples were boiled in a water bath for 40 min, cooled and
centrifuged at 3,000 rpm for 15 min. Then, the liquid supernatants were
collected. This extraction procedure was repeated twice, finally the
supernatants were combined and raised to a set volume of 10 mL. Determination of NO3−,
H2PO4−, SO42−,
C2O42− and Cl− in the
supernatant by means of ion chromatography (DX-300ion chromatographic system,
AS4A-SC chromatographic column, CDM-II electrical conductivity detector, mobile
phase: Na2CO3/NaHCO3 = 1.7/1.8 mM, Dionex, Sunnyvale, CA, USA). The
concentrations of Na+, Mg2+, Ca2+, K+,
P5+, Fe3+, B3+, Zn2+, Cu2+and
Mn2+ were measured with an atomic absorption spectrophotometer
(Super 990F, Beijing Purkinje General Instrument Co. Ltd., Beijing, China).
Each sample was measured in triplicate (Yang et al. 2017).
Determination of key enzymatic
activity levels
Determination of the nitrate reductase (NR) activity level: The sulfamate colorimetric method was used to
determine the activity level of NR (Li et
al. 2019). Add a reaction mixture containing 200 mM KNO3, 5 mM EDTA and
0.15 mM NADH to 100 mM phosphate buffer pH 7.5, incubate at
30°C for 1 h, then add 2 mL sulfate and 2 mL α-naphthylamine. Measure the
absorbance at 540 nm. Enzyme activity is expressed in µmol·min-1.
Determination
of the glutamine synthetase (GS) activity level: The GS enzyme extract was prepared
as follows: 1g sample was weighed, placed in liquid nitrogen and ground. Then,
8 mL of extractive solution (100 mM
Tris-HCl, 0.5 mM EDTA and 5 mM β-Mercaptoethanol, pH 7.5) was
added, and the sample was filtered through two layers of gauze, followed by
centrifugation at 4°C for 15,000 r·min−1 for 20 min. The supernatant was
used for enzymatic activity determination.
Enzyme activity determination: 1.6 mL reaction solution, which contains
50 mM hydrochloric acid buffer
solution pH (7.0), sodium glutamate solution 0.3 M pH (7.0), 0.3 M MgSO4
and 30 mM ATP Solution-Na (pH 7.0),
then It is 0.6 mL enzyme extract solution (CK immediately accepts 1.0 mL FeCl3
reagent). Keep the reaction solution in a water bath at 25°C for 5 min, add 0.2
mL of hydroxylamine reagent to start the reaction, and continue in a water bath
at 25°C for 15 min. Then add 1 mL of FeCl3 reagent to stop the
reaction. The mixed solution was centrifuged at 15,000 rpm for 10 min and the
optical density of the supernatant was measured at 540 nm (Rhodes et al. 1975).
Determination of the aspartate aminotransferase
(GOT) activity level: To determine the GOT level, two test tubes were
used, one as the sample tube, to which 0.1 mL of crude enzyme preparation and
0.5 mL of GOT substrate solution was added and the other was the CK tube, to
which only 0.1 mL of crude enzyme preparation was added. Both tubes were placed
in a 37°C water bath at the same time. Take it out
after 30 min, add 0.5 mL of 2,4-dinitrophenylhydrazine solution to each tube to
stop the reaction and add 0.5 mL of GOT substrate solution to the CK tube. The
two tubes were then placed in a 37°C water bath for
20 min and 5.0 mL of 0.4 mol·L−1NaOH was added to each tube
and mixed. After 10 min, a spectrophotometer was used to measure the absorbance
at 500nm after distilled water was adjusted to read zero (Wu et al. 1998).
Determination of the alanine aminotransferase (GPT)
activity level: The determination of GPT activity was assessed by determining the
reduction in absorbance owing to the consumption of NADH at 340 nm. The
reaction buffer was composed of 15 mM
a-oxoglutarate, 0.15 mM NADH, 0.5 M 1-alanine, and 5-unit lactate
dehydrogenase in 50 mM Tris HCl
buffer (pH 7.5). Then, 100 µL enzyme
was added to initiate the extraction reaction (Ullah et al. 2019).
Determination of glutamate dehydrogenase (GDH) activity:
A reaction mixture of 23.1 mM α-ketoglutarate, 231 mM NH4Cl, 30 mM CaCl2 and 6 mM NADH was used to determine the
activity of NADH-GDH in 100 mM
Tris-HCl buffer (pH 8.0). The reaction starts with the addition of the enzyme
extract. The NADH-GDH reaction mixture consists of 100 mM 1-glutamic acid, 1 mM
NAD in 100 mM Tris-HCl buffer (pH
8.8) and enzyme extract. The oxidizing activity of NADH (NADH-GDH) and the
reducing activity of NADH (NADH-GDH) were measured by spectrophotometry.
Enzymatic activity was expressed in units of μmol·g−1 protein (Loulakakis
and Roubelakis-Angelakis 1991).
Determination of glutamate synthase (GOGAT) activity:
The extraction solution was
prepared in the same way as GS. Determination of enzymatic activity: the total
volume of the reaction system was 3 mL (containing 0.4 mL 20 mM L-glutamine, 0.05 mL 0.1M α-ketoglutarate, 0.1 mL 10 mM KCl, 0.1 mL 3 mM NADH and 0.3 mL enzyme solution), an insufficient volume was made
up with 25 mM Tris-HCl at pH 7.6. The
reaction is initiated by L-glutamine and 10 successive absorbance values are
measured at 340 nm every 30 sec. The segment with a stable decrease in optical
density was used to measure enzymatic activity (Singh and Srivastava 1986).
The data were organized using Microsoft Excel
2007. The data values are presented as means ± SE. The data were analyzed
statistically using a two-way analysis of variance in SPSS (Version 13.0, SPSS,
Chicago, IL, USA) and significant differences among treatment means were
detected at P < 0.05 in accordance
with Duncan’s method. SigmaPlot 10.0 was used to construct the graphics.
Results
Changes in plant growth and total
C and N contents
Under LN-stress
conditions, the plant heights, Up and Under FWs and Up and
Under DWs of cultivated soybean decreased, that correlated with the
increase in the LN stress’ intensity (Table 2).
Under high-intensity stress conditions, Table 1: Chemical composition of CK, N1, N2 & N3
|
Characteristics |
Chemical
reagents |
10 L
(1/2) |
10 L
(1/3) |
10 L
(1/4) |
Total mass (g) |
|
|
|
Weighed
mass (g) |
Weighed
mass (g) |
Weighed
mass (g) |
|
|
1 |
Ca(NO3)2.4H2O |
41.035 |
27.360 |
20.520 |
88.910 |
|
2 |
MgSO4.7H2O |
61.620 |
61.620 |
61.620 |
184.860 |
|
3 |
KH2PO4 |
27.220 |
27.220 |
27.220 |
81.660 |
|
4 |
KNO3 |
25.280 |
16.850 |
12.640 |
54.770 |
|
5 |
Na-EDTA |
7.450 |
7.450 |
7.450 |
22.350 |
|
6 |
FeSO4.7H2O |
5.570 |
5.570 |
5.570 |
16.710 |
|
|
H3BO3 |
2.860 |
2.860 |
2.860 |
8.580 |
|
|
MnSO4 |
1.015 |
1.015 |
1.015 |
3.045 |
|
|
CuSO4.5H2O |
0.079 |
0.079 |
0.079 |
0.237 |
|
|
ZnSO4.7H2O |
0.220 |
0.220 |
0.220 |
0.660 |
|
Substitute reagent |
H2MoO4 |
0.090 |
0.090 |
0.090 |
0.270 |
|
Ca(NO3)2.4H2O |
|
|
|
|
|
|
KNO3 |
CaCl2.2H2O |
25.550 |
34.060 |
38.320 |
97.930 |
Preparation of low nitrogen stress solution (10 times solution)
Common Hoagland
Nutrient Solution Formula
|
Number |
Name of drug |
100 times
mother liquor (g·L-1) |
1 times
the amount (mL·L-1) |
100 times
mother liquor (g·L-1) |
1 times
the amount (mL·L-1) |
|
1 |
Ca(NO3)2 |
82.07 g |
10 |
410.35g |
2 |
|
2 |
MgSO4·7H2O |
61.62 g |
10 |
308.10g |
2 |
|
3 |
KH2PO4 |
27.22 g |
10 |
136.10g |
2 |
|
KNO3 |
50.56 g |
10 |
252.80g |
2 |
|
|
4 |
Fe-EDTA |
Na-EDTA 7.45 g; FeSO4·7H2O 5.57 g |
1 |
||
|
5 |
Trace
elements |
H3BO3
2.860 g; MnSO4
1.015 g; CuSO4·5H2O
0.079 g; ZnSO4·7H2O
0.220 g; H2MoO4 0.090 g |
1 |
||
Table 2: Biomass changes of wild
soybean and cultivated soybean under control and three different intensities of
low nitrogen stress
|
Soybeans |
Growth
parameters |
Treatments |
|
|
|
|||
|
|
|
CK |
N1 |
N2 |
N3 |
log2(N1/CK) |
log2(N2/CK) |
log2(N3/Ck) |
|
W |
Shoot height
(cm) |
120.00 ± 10.00 |
135.00 ± 20.21 |
130.33 ± 4.67 |
115.00 ± 43.00 |
0.17 |
0.12 |
-0.15 |
|
Root
length (cm) |
30.50 ± 2.50 |
33.67 ± 0.88 |
38.67 ± 4.70 |
51.00 ± 1.50 |
0.14 |
0.34 |
0.74* |
|
|
Fresh
weight of shoots (g) |
57.50 ± 1.90 |
55.33 ± 4.05 |
51.93 ± 3.20 |
50.55 ± 11.35 |
-0.06 |
-0.15 |
-0.54 |
|
|
Dry
weight of shoots (g) |
9.98 ± 0.40 |
9.24 ± 0.70 |
8.98 ± 0.71 |
7.28 ± 0.20 |
-0.11 |
-0.15 |
-1.22** |
|
|
Fresh
weight of roots (g) |
13.80 ± 1.50 |
13.33 ± 1.62 |
15.83 ± 1.33 |
12.15 ± 1.95 |
-0.05 |
0.20 |
-0.18 |
|
|
Dry
weight of roots (g) |
1.37 ± 0.26 |
1.13 ± 0.12 |
1.25 ± 0.15 |
1.07 ± 0.19 |
-0.27 |
-0.14 |
-0.36 |
|
|
M |
Shoot height
(cm) |
59.50 ± 1.50 |
54.30 ± 2.30 |
61.70 ± 4.90 |
51.00 ± 5.00 |
-0.13 |
0.05 |
-0.17 |
|
Root
length (cm) |
34.50 ± 0.50 |
32.67 ± 3.93 |
34.00 ± 5.57 |
28.00 ± 3.00 |
-0.08 |
-0.02 |
-0.30 |
|
|
Fresh
weight of shoots (g) |
65.30 ± 13.60 |
69.93 ± 3.17 |
75.27 ± 5.78 |
56.60 ± 6.90 |
0.10 |
0.21 |
-0.21 |
|
|
Dry
weight of shoots (g) |
11.53 ± 2.76 |
11.55 ± 0.97 |
13.05 ± 1.22 |
6.50 ± 1.57 |
0.002 |
0.18 |
-0.14 |
|
|
Fresh
weight of roots (g) |
23.55 ± 4.85 |
21.00 ± 1.15 |
21.33 ± 1.42 |
18.00 ± 1.30 |
-0.17 |
-0.14 |
-0.09 |
|
|
Dry
weight of roots (g) |
3.43 ± 0.74 |
2.62 ± 0.11 |
2.79 ± 0.34 |
2.80 ± 0.41 |
-0.39 |
-0.30 |
-0.29 |
|
decrease was by 10.92, 9.00,
13.26, 6.14 and 10.83%, respectively; however, the decreasing trends in plant
heights, Up and Under FWs, and Up and Under DWs of wild soybean did not reach a
significant level (P > 0.05) and
as the LN-stress intensity increased, there were no significant differences among
the treated tissues. The root length of wild soybean increased significantly (P < 0.05) and the root-shoot ratio
showed an increasing trend, but there were no such
increasing trends in cultivated soybean. Compared to CK, there was a slight
increase in the nodule weights in the two soybean genotype groups, but the
difference was not significant (P >
0.05). Under stress, the contents of C and N in the
young and old leaves of the two genotypes of soybean were lower than CK (Table
3). In addition, as the LN stress intensity increased, the C and N contents
decreased gradually. Under high-intensity stress conditions, the C contents of
wild soybean young and old leaves decreased by 6.06 and 18.93%, respectively, and
the N contents decreased by 54.4 and 31.2%, respectively. The C contents of
cultivated soybean young and old leaves decreased by 39.53 and 23.84%,
respectively, and the N contents decreased by 55.5 and 39.6%, respectively. Compared with the old leaves, the decrease in the N
content of the young leaves was greater. As the LN-stress intensity increased,
the degrees of C and N decreases in wild soybean was less than in cultivated
soybean.
Changes in photosynthetic traits
As the stress intensity increased, PN
and Cr values in young and old leaves of cultivated soybean showed decreasing
trends (Fig. 1). Under high-intensity stress conditions, compared with the CK,
the PN values of cultivated soybean young and old leaves decreased significantly (P < 0.05), while in wild soybean
young and old leaves also decreased, but not significantly (P > 0.05). Among the three
different intensity LN-stress treatments, Cr values in cultivated
soybean young and old leaves decreased significantly (P < 0.05), but not in wild soybean young leaves (P > 0.05). Gs and E values increased
in wild soybean young leaves, but decreased in wild
soybean old leaves. The Gs and E values of the young leaves and old leaves of
the cultivated soybean plant showed a downward trend. With the increase of
stress intensity, the Ci of wild soybean young and old leaves increased, while
the Ci of cultivated soybean leaves decreased. As the LN-stress intensity
increased, the Chl a,
Chl b, and Chl (a+b) contents in the
young and old leaves of the two soybean genotypes showed gradual decreases
compared with the CK. Under high-intensity stress conditions, the Chl a, Chl b and Chl (a+b) contents in wild
soybean young leaves decreased by 25, 32.4 and 27.5%, respectively, while in
the old leaves they decreased by 36.8, 47.1 and 39.9%, respectively, compared
with the CK. Correspondingly, their contents in cultivated soybean young leaves
decreased by 33.72, 31.34 and 33.05%, respectively, while in the old leaves they
decreased by 56.10, 45.32 and 53.47%, respectively. Compared with the CK, the
Chl a, Chl b and Chl (a+b) contents decreased more in the old leaves than in
the young leaves. As the LN-stress intensity increased, the Car content showed
decreased in both young and old leaves of cultivated soybean and under high-intensity stress, the Car contents of
cultivated soybean young and old leaves decreased significantly (P < 0.05).
Table 3: Carbon and nitrogen contents in young and old leaves of
two soybean varieties under control and three different intensities of low
nitrogen stress
|
Leaf
age |
Total content (%) |
Fold changes
Log2 (N1/CK) |
Fold changes
Log2(N2/CK) |
Fold changes
Log2(N3/CK) |
|||||||||||
|
M |
|
|
|
W |
|
|
|
|
|
|
|
|
|
||
|
CK |
N1 |
N2 |
N3 |
CK |
N1 |
N2 |
N3 |
M |
W |
M |
W |
M |
W |
||
|
YL |
Nitrogen |
4.49 ± 0.11 |
3.13±0.09 |
4.67±0.06 |
1.99±0.16 |
4.74±0.08 |
2.47±0.17 |
4.19±0.06 |
2.08±0.07 |
-0.52** |
-0.94** |
0.06 |
-0.18** |
-1.17** |
-1.19** |
|
|
Carbon |
41.61±0.81 |
36.18±0.33 |
44.39±0.25 |
39.09±1.38 |
41.28±0.58 |
22.83±1.72 |
40.74±0.34 |
24.96±0.56 |
-0.20** |
-0.85** |
0.10* |
-0.02 |
-0.09 |
-0.73** |
|
|
C/N |
9.27 |
11.57 |
9.500 |
19.58 |
8.71 |
9.22 |
9.72 |
11.99 |
0.32 |
0.08 |
0.04 |
0.16 |
1.08 |
0.38 |
|
OL |
Nitrogen |
3.82±0.04 |
2.01±0.25 |
3.65±0.11 |
2.31±0.11 |
3.44±0.07 |
2.54±0.05 |
3.76±0.05 |
2.37±0.06 |
-0.93** |
-0.44** |
-0.07* |
0.13* |
-0.73** |
-0.54** |
|
|
Carbon |
42.80±0.03 |
39.01±1.83 |
42.24±0.24 |
34.84±0.19 |
40.62±0.40 |
32.19±0.62 |
41.52±0.11 |
30.93±0.67 |
-0.13 |
-0.34** |
-0.02 |
0.03 |
-0.30** |
-0.40** |
|
|
C/N |
11.19 |
19.40 |
11.59 |
15.08 |
11.81 |
12.69 |
11.05 |
13.08 |
0.80 |
0.10 |
0.05 |
-0.10 |
0.43 |
0.15 |
%20321-331_files/image002.jpg)
Fig. 1: The changes in photosynthetic characteristics in
young and old leaves from wild and cultivated soybean
under control and N deficiency
(A) pN:
net photosynthetic rate, (B) gs: stomatal
conductance, (C) E: transpiration rate, (D) Ci: CO2 concentration
under stomata, (E) Cr: atmospheric CO2 concentrations, (F) Chl a:
chlorophyll a, (G) Chl b:
Chlorophyll b, (H) Chl a+b: chlorophyll a +
chlorophyll b, (I) Car: carotenoid, *and ** indicate significant (P < 0.05) and highly significant (P < 0.01) differences
Ion content changes
With the increase in the LN-stress intensity,
the Na+ content of the young cultivated soybean leaves decreased
significantly (P < 0.05) and the
Na+ content of the young wild soybean leaves and old leaves
increased significantly (Table 4). Under high-intensity
stress, the Na+ content in cultivated soybean young leaves
significantly (P < 0.01) decreased
compared with the CK group, but wild soybean is not like that. The K+ contents
decreased significantly in wild soybean young and old leaves under high-intensity stress conditions (P < 0.05). The SO42− content
decreased in wild soybean young leaves, increased in wild soybean old leaves, but increased
in both cultivated soybean young and old leaves. Compared with the CK, the P5+,
PO42− and B3+contents
increased and the Mg2+ and the NO3−
content in the young and old leaves of the two genotypes of soybeans showed a
downward trend. P5+ content in wild soybean young leaves increased
significantly under high-intensity LN stress (P < 0.05), but there was no such phenomenon in cultivated soybean.
The growth of young leaves of B3+ is much higher than that of
cultivated soybean. The content of NO3− wild
soybean in young leaves and old leaves was lower than cultivated soybean, and
young leaves were lower than old leaves. The decrease in Mg2+ content
in young wild soybean leaves was less than that in cultivated soybean young
leaves. As the stress intensity increased, the C2O4−contents
in wild soybean and cultivated soybean young leaves increased, and under
high-intensity stress conditions, the C2O4− content
in wild soybean young leaves showed a significant increase compared with the
CK. With the increase of stress intensity, the content of Fe3+, Mn2+
and Zn2+ in wild soybean leaves was significantly higher than that
in cultivated soybean leaves. More useful ions are accumulated in new leaves
than in old leaves. As the stress intensity increased, the extent of the ion
content decrease in cultivated soybean was greater than in wild soybean.
Changes in the key N metabolism-related
enzymatic activities
As the LN-stress intensity increased, the NR and
GOT activities increased in wild soybean young leaves,
but decreased in cultivated soybean young leaves, compared with CK (Fig. 2). NR
and GPT activities increased in wild soybean young
leaves, but decreased in old leaves. The NR, GS and GOT activities decreased in
cultivated soybean young and old leaves. The GPT activity increased in
cultivated soybean young leaves, but there was no significant change in old
leaves (P > 0.05).
The level of increase in GPT activity was greater in wild soybean than in
cultivated soybean young leaves. The GDH activities decreased in the young and
old leaves of the two soybean genotypes, but the changes were not significant (P > 0.05). GOGAT activity in wild
soybean young leaves decreased, while GOGAT activity increased in wild soybean
old leaves and GOGAT activity increased in cultivated soybean young leaves and
old leaves. In the two soybean genotypes, the NR activities in the young leaves
were significantly higher than in the old leaves. The activities of NR and GS
in old leaves of two soybean genotypes were significantly reduced under
high-intensity stress (P < 0.01).
The GOT activity of cultivated soybean young leaves is significantly reduced (P < 0.05).
The GPT activity significantly increased in wild soybean young leaves (P < 0.05), but not in cultivated
soybean young leaves. As the stress intensity increased, the key enzymes
maintained higher activities in the young leaves compared with the old leaves.
As the LN stress intensity increased, the activities of these key enzymes were
higher in wild soybean than that in cultivated soybean.
Discussion
N is an essential nutrient for crop growth and
development, and LN stress can inhibit plant
development (Boussadia et al.
2010). This experiment showed that
the growth rates and biomasses of two soybean
genotypes were severely inhibited under different degrees of LN stress,
especially in cultivated soybean. The root length of wild soybean increased,
indicating that wild soybean can resist the LN stress. The
number of nodules in the two genotypes of soybean seedlings is very limited and
the nodule weight does not change significantly under the conditions of CK and
LN, which is in agreement with previous studies (Li et al. 2018). The total C content (%) and total N content (%)
showed that cultivated soybean young and old leaves had a worse nitrogen and N
deficiencies than wild soybean, confirming that the latter had a stronger
tolerance to LN stress than cultivated soybean. These results confirmed that
wild soybean was more tolerant to LN stress than cultivated soybean.
The photosynthetic capacity of a leaf is related
to the N content, mainly because the proteins and thylakoids in the Calvin
cycle form the majority of N present in leaves (Evans 1989). However, the photosynthesis of crops can be
reduced under LN-stress conditions (Cechin and Fumis 2004). Here, under different
degrees of LN stress, PN decreased in the two soybean genotypes,
especially under high-intensity stress. The PN values decreased in
the young and old leaves of cultivated soybean, indicating that its inhibition
in cultivated soybean is greater under LN-stress conditions. Both gs
and Ci are related to plant transpiration intensity, stomatal opening, and the
ability of mesophyll cells to assimilate CO2 (Mohamed et al. 2017). In this
experiment, the change trends of Ci and gs were consistent, with
both decreasing in cultivated soybean young and old leaves. The PN
in cultivated soybean decreased as a result of stomatal limitation. The change
trends of gs and Ci in wild soybean old leaves were inconsistent,
with gs decreasing and Ci increasing, which indicated that the
decrease in wild soybean’s PN was the result of nonstomatal factors.
The increase of E in wild soybean young leaves promoted the transport of water
and inorganic salts, as well as the migration and transportation of ions (Li et al. 2019). The Chl a, Chl b, and Chl (a+b) contents decreased as the LN-stress intensity
increased. The Chl contents of the two soybean genotypes decreased
under different degrees of LN stress, and the Chl contents in wild soybean
young and old leaves decreased less than in cultivated soybean. A decrease in
the photosynthetic pigment content can cause a decrease in PN, which
indicates that the inhibition of wild soybean’s PN was weaker under
LN-stress conditions. Between young and old leaves, the extent of the Chl content’s
decrease was greater in the latter, indicating that LN stress inhibited the PN
levels of old leaves to a greater extent than young leaves. Wild soybean can increase Car
accumulation to resist abiotic stress (Li et al. 2018). Here, the Car contents
decreased significantly in cultivated soybean young and old leaves (P < 0.05) and the contents also
decreased in wild soybean young and old leaves, but not significantly (P > 0.05), which corroborated
previous results. Thus, the inhibitory effect of LN stress on the light
contracting function was stronger in cultivated soybean than wild soybean.
An insufficient N supply leads
to a reduction in amino acids, proteins and other N-containing compounds,
disrupting multiple energy and substances’ metabolic pathways. This results in
changes in plant energy supply and multiple nutrient transporter activities,
ultimately leading to changes in the absorption and distribution of nutrient Table 4: Ion contents in young and old leaves of two soybean genotypes under
control and three different intensities of low nitrogen stress
|
Young leaves |
concentration
(mmol. L− 1 ) |
Fold changes |
||||||||||||
|
M |
W |
Log2
(N1/CK) |
Log2(N2/CK) |
Log2(N3/CK) |
||||||||||
|
|
CK |
N1 |
N2 |
N3 |
CK |
N1 |
N2 |
N3 |
M |
W |
M |
W |
M |
W |
|
NO3− |
2.78 ± 0.00 |
3.62 ± 0.09 |
3.26 ± 0.08 |
1.29 ± 0.00 |
2.26 ± 0.11 |
5.94 ± 0.29 |
3.16 ± 0.08 |
1.40 ± 0.01 |
0.38** |
1.40** |
0.23** |
0.49** |
-1.10** |
-0.69** |
|
H2PO4− |
1.62 ± 1.53 |
1.53 ± 0.06 |
1.90 ± 0.02 |
2.46 ± 0.30 |
1.86 ± 0.26 |
1.95 ± 0.11 |
1.82 ± 0.04 |
3.20 ± 0.97 |
-0.09** |
0.07** |
0.23** |
-0.03** |
0.60 |
0.78** |
|
SO42− |
1.73 ± 0.42 |
2.15 ± 0.08 |
1.20 ± 0.03 |
1.81 ± 2.25 |
2.61 ± 0.20 |
2.54 ± 0.00 |
1.81 ± 0.04 |
2.36 ± 0.70 |
0.31** |
-0.04** |
-0.52** |
-0.53** |
0.07 |
-0.15 |
|
C2O42− |
1.37 ± 0.02 |
0.73 ± 0.00 |
0.43 ± 0.00 |
1.47 ± 0.03 |
1.03 ± 0.06 |
0.57 ± 0.02 |
0.54 ± 0.00 |
1.72 ± 0.02 |
-0.91** |
-0.85** |
-1.66** |
-0.92** |
0.09 |
0.74** |
|
Cl− |
1.28 ± 0.01 |
0.88 ± 0.02 |
1.82 ± 0.05 |
1.84 ± 0.02 |
2.43 ± 0.13 |
1.73 ± 0.34 |
2.03 ± 0.02 |
4.47 ± 0.08 |
-0.54 |
-0.49 |
0.51 |
-0.25 |
0.52 |
0.88 |
|
Na+ |
2.79 ± 0.01 |
2.12 ± 0.04 |
2.27 ± 1.13 |
1.52 ± 0.06 |
2.15 ± 0.15 |
2.19 ± 0.03 |
2.26 ± 0.00 |
2.05 ± 0.05 |
-0.39** |
0.03 |
-0.30 |
0.08 |
-0.87** |
0.06 |
|
Mg2+ |
24.63
± 1.29 |
19.89
± 0.97 |
19.72
± 7.24 |
20.39
± 0.31 |
28.30
± 0.79 |
23.42
± 0.08 |
23.56
± 0.10 |
26.48
± 0.13 |
-0.31 |
-0.27 |
-0.32 |
-0.26 |
-0.27 |
-0.10 |
|
Mn2+ |
0.14 ± 0.01 |
0.15 ± 0.01 |
0.23 ± 0.00 |
0.12 ± 0.00 |
0.09 ± 0.00 |
0.13 ± 0.01 |
0.14 ± 0.00 |
0.13 ± 0.00 |
0.18 |
0.52 |
0.76 |
0.63 |
-0.15 |
0.59** |
|
B3+ |
0.33 ± 0.00 |
0.54 ± 0.03 |
0.88 ± 0.11 |
0.69 ± 0.01 |
0.35 ± 0.01 |
1.68 ± 0.02 |
1.39 ± 0.01 |
1.62 ± 0.00 |
0.71** |
2.15 |
1.41** |
1.87 |
1.05** |
2.22** |
|
Fe3+ |
0.06 ± 0.00 |
0.06 ± 0.01 |
0.13 ± 0.01 |
0.08 ± 0.00 |
0.05 ± 0.00 |
0.08 ± 0.00 |
0.03 ± 0.00 |
0.12 ± 0.00 |
1.00 |
0.49 |
1.12 |
0.58 |
0.43* |
1.17** |
|
Ca2+ |
11.66
± 0.39 |
10.06
± 0.44 |
13.80
± 0.83 |
15.41
± 0.14 |
14.60
± 0.41 |
11.92
± 0.02 |
12.79
± 0.10 |
14.00
± 0.05 |
-0.21 |
-0.29 |
0.24* |
-0.19 |
0.40** |
-0.06 |
|
K+ |
144.34
± 4.49 |
134.37
± 4.34 |
146.62
±9.79 |
152.64±2.08 |
191.66±5.18 |
190.22±1.11 |
168.46±1.45 |
169.57±0.30 |
-0.10 |
-0.01 |
0.02 |
-0.19 |
0.08 |
-0.18* |
|
P5+ |
35.01
± 1.78 |
31.37
± 0.19 |
35.97
± 2.30 |
39.01
± 0.38 |
41.81
± 0.68 |
48.61
± 0.23 |
49.90
± 0.07 |
57.06
± 0.13 |
-0.16 |
0.22 |
0.04 |
0.26 |
0.16 |
0.45** |
|
Zn2+ |
0.22 ± 0.21 |
0.23 ± 0.24 |
0.23 ± 0.28 |
0.24 ± 0.29 |
0.14 ± 0.01 |
0.28 ± 0.00 |
0.23 ± 0.00 |
0.24 ± 0.00 |
0.06 |
1.06 |
0.06 |
0.78 |
0.13 |
0.78 |
|
NO3− |
2.78 ± 0.00 |
3.62 ± 0.09 |
3.26 ± 0.08 |
1.29 ± 0.00 |
2.26 ± 0.11 |
5.94 ± 0.29 |
3.16 ± 0.08 |
1.40 ± 0.01 |
0.38** |
1.40** |
0.23** |
0.49** |
-1.10** |
-0.69** |
|
H2PO4− |
1.62 ± 1.53 |
1.53 ± 0.06 |
1.90 ± 0.02 |
2.46 ± 0.30 |
1.86 ± 0.26 |
1.95 ± 0.11 |
1.82 ± 0.04 |
3.20 ± 0.97 |
-0.09** |
0.07** |
0.23** |
-0.03** |
0.60 |
0.78** |
|
SO42− |
1.73 ± 0.42 |
2.15 ± 0.08 |
1.20 ± 0.03 |
1.81 ± 2.25 |
2.61 ± 0.20 |
2.54 ± 0.00 |
1.81 ± 0.04 |
2.36 ± 0.70 |
0.31** |
-0.04** |
-0.52** |
-0.53** |
0.07 |
-0.15 |
|
Old leaves |
concentration
(mmol. L− 1 ) |
Fold changes |
||||||||||||
|
M |
W |
Log2
(N1/CK) |
Log2(N2/CK) |
Log2(N3/CK) |
||||||||||
|
|
CK |
N1 |
N2 |
N3 |
CK |
N1 |
N2 |
N3 |
M |
W |
M |
W |
M |
W |
|
NO3− |
4.94 ± 0.04 |
6.72 ± 0.14 |
2.93 ± 0.03 |
1.08 ± 0.04 |
3.96 ± 0.04 |
2.21 ± 0.14 |
1.25 ± 0.00 |
0.92 ± 0.04 |
0.44** |
-0.84** |
-0.75** |
-1.66** |
-2.19** |
-2.10** |
|
H2PO4− |
2.00 ± 0.14 |
1.49 ± 0.02 |
1.70 ± 0.00 |
2.73 ± 0.66 |
2.06 ± 0.04 |
1.90 ± 0.15 |
2.56 ± 0.08 |
4.60±0.66** |
-0.43** |
-0.11** |
-0.24** |
0.32** |
0.45** |
1.16** |
|
SO42− |
1.23 ± 0.15 |
3.03 ± 0.03 |
2.33 ± 0.02 |
2.80 ± 0.11 |
2.16 ± 0.01 |
3.33 ± 0.27 |
2.35 ± 0.04 |
2.79 ± 0.11 |
1.30** |
0.63* |
0.93** |
0.13* |
1.19** |
0.37* |
|
C2O42− |
1.05 ±0.04 |
0.51 ± 0.05 |
0.44 ± 0.11 |
1.14 ± 0.04 |
1.33 ± 0.02 |
0.46 ± 0.01 |
0.77 ± 0.00 |
1.34 ± 0.04 |
-1.03** |
-1.52** |
-1.24** |
-0.79** |
0.12 |
0.01 |
|
Cl− |
2.25 ± 0.01 |
2.31 ± 0.02 |
3.06 ± 0.09 |
3.10 ± 0.04 |
2.49 ± 0.21 |
3.21 ± 0.22 |
3.43 ± 0.05 |
7.77 ± 0.28 |
0.04 |
0.37 |
0.44 |
0.46 |
0.46 |
1.64 |
|
Na+ |
1.03 ± 0.03 |
1.56 ± 0.16 |
1.62 ± 0.01 |
2.40 ± 0.06 |
1.01 ± 0.05 |
2.05 ± 0.07 |
3.88 ± 0.75 |
1.82 ± 0.06 |
0.60* |
1.02 |
0.66** |
1.94 |
1.22 |
0.85 |
|
Mg2+ |
45.05
± 0.75 |
40.76
± 0.75 |
34.88
± 1.54 |
38.95
± 0.18 |
46.4 ± 0.12 |
37.90
± 0.20 |
30.04
± 0.68 |
33.28
± 0.18 |
-0.14* |
-0.29 |
-0.37** |
-0.63 |
-0.21* |
-0.48 |
|
Mn2+ |
0.17 ± 0.01 |
0.23 ± 0.00 |
0.22 ± 0.01 |
0.19 ± 0.04 |
0.12 ± 0.00 |
0.21 ± 0.01 |
0.20 ± 0.01 |
0.16 ± 0.04 |
0.47** |
0.83 |
0.41** |
0.77 |
0.21 |
0.46 |
|
B3+ |
0.86 ± 0.02 |
3.31 ± 0.07 |
4.15 ± 0.16 |
5.21 ± 0.05 |
1.09 ± 0.03 |
5.74 ± 0.10 |
4.79 ± 0.06 |
4.86 ± 0.05 |
1.94** |
2.40 |
2.27** |
2.14 |
2.60** |
2.16 |
|
Fe3+ |
0.10 ± 0.03 |
0.07 ± 0.00 |
0.07 ± 0.01 |
0.09 ± 0.00 |
0.04 ± 0.00 |
0.04 ± 0.00 |
0.05 ± 0.00 |
0.09 ± 0.00 |
-0.52 |
0.02 |
-0.53 |
0.34 |
-0.15 |
0.98 |
|
Ca2+ |
59.14
± 0.55 |
59.24
± 0.40 |
44.15
± 1.13 |
48.22
± 0.07 |
48.96
± 0.14 |
43.87
± 0.47 |
27.27
± 0.90 |
31.32
± 0.07 |
0.01 |
-0.16 |
-0.42** |
-0.84 |
-0.29** |
-0.64 |
|
K+ |
152.58
± 2.48 |
149.48
± 3.00 |
150.37
± 7.24 |
139.95
± 0.70 |
163.44
± 0.07 |
170.07±0.09 |
162.14±5.60 |
172.59±0.07 |
-0.03 |
0.06 |
-0.02 |
-0.01 |
-0.12 |
0.08 |
|
P5+ |
27.36
± 0.05 |
26.85
± 0.14 |
33.38
± 0.83 |
47.0 8
±0.04 |
32.87
± 0.12 |
36.64
± 0.88 |
52.92
± 2.30 |
70.81
± 0.04 |
-0.03* |
0.16 |
0.29** |
0.69 |
0.78** |
1.11 |
|
Zn2+ |
0.18 ± 0.20 |
0.19 ± 0.20 |
0.22 ± 0.24 |
0.24 ± 0.22 |
0.09 ± 0.00 |
0.26 ± 0.00 |
0.26 ± 0.01 |
0.35 ± 0.00 |
0.08 |
1.48 |
0.29 |
1.51 |
0.42 |
1.93 |
|
NO3− |
2.78 ± 0.00 |
3.62 ± 0.09 |
3.26 ± 0.08 |
1.29 ± 0.00 |
2.26 ± 0.11 |
5.94 ± 0.29 |
3.16 ± 0.08 |
1.40 ± 0.01 |
0.38** |
1.40** |
0.23** |
0.49** |
-1.10** |
-0.69** |
|
H2PO4− |
1.62 ± 1.53 |
1.53 ± 0.06 |
1.90 ± 0.02 |
2.46 ± 0.30 |
1.86 ± 0.26 |
1.95 ± 0.11 |
1.82 ± 0.04 |
3.20 ± 0.97 |
-0.09** |
0.07** |
0.23** |
-0.03** |
0.60 |
0.78** |
|
SO42− |
1.73 ± 0.42 |
2.15 ± 0.08 |
1.20 ± 0.03 |
1.81 ± 2.25 |
2.61 ± 0.20 |
2.54 ± 0.00 |
1.81 ± 0.04 |
2.36 ± 0.70 |
0.31** |
-0.04** |
-0.52** |
-0.53** |
0.07 |
-0.15 |
%20321-331_files/image004.jpg)
Fig. 2: The changes in nitrogen metabolism enzyme activities
in young and old leaves from wild and cultivated soybean under control and N
deficiency
(A) NR: nitrate reductase, (C)
GS: glutamine synthetase, (E) GOT:
aspartate aminotransferase, (B) NR:
nitrate reductase, (D) GS: glutamine
synthetase, (F) GOT: aspartate
aminotransferase, (G) GPT: alanine
aminotransferase, (I) GDH: glutamate
dehydrogenase, (K) GOGAT: glutamate
synthase, (H) GPT: alanine
aminotransferase, (J) GDH: glutamate
dehydrogenase, (L) GOGAT: glutamate
synthase. *and ** indicate significant (P
< 0.05) and highly significant (P
< 0.01) differences, respectively
elements (Quan et al.
2016). In this study, under LN-stress conditions, especially
high-intensity stress, the P5+ content increased in the young and
old leaves of the two soybean genotypes, the Na+ content increased
in wild soybean young leaves, but decreased significantly in cultivated soybean
young leaves, the K+ content decreased significantly in young and
old leaves of wild soybean but increased in young leaves of cultivated soybean
and the Mg2+ content of young soybean leaves of the two genotypes all showed a
downward trend and the Mg2+ content of
young soybean leaves of cultivated soybean type soybean decreased more obviously. Compared with the control, the content of Fe3+,
Mn2+, B3+ and Zn2+ in wild soybean young
leaves was significantly higher than that in cultivated soybean young leaves. K+
participates in very important metabolic activities in plants and plays
important roles in regulating ion balance and in maintaining cell turgor,
ribosome function, and protein synthesis (Fan et al. 2009). Na+ can inhibit K+
absorption (Li et al. 2019). In this
experiment, as the LN-stress intensity increased, Na+ accumulated,
but the K+ content decreased in wild soybean young and old leaves.
However, the K+ content was much greater than the Na+
content, indicating that wild soybean maintained a high K+ /Na+
value to resist abiotic stress, which was consistent with a previous study (Li et al. 2019). Here, the K+ content
in cultivated soybean young leaves increased, but it decreased in the old
leaves, which indicated that K+ can be transferred from the
cultivated soybean old leaves to young leaves, thereby regulating their ion
balance and maintaining the growth and development of young leaves. Wild
soybean maintained a certain Mg2+ content to produce more
photosynthetic pigments, which reduces the damage to photosynthesis (Han et al. 2013). Fe3+
is a carrier in the electron transport of photosynthesis, and it participates
in photosynthesis and N fixation, protecting the photosynthetic apparatus and
maintaining normal growth (Cakmak 2000). Mn2+ as an
enzyme activator, participates in many metabolic processes by regulating
enzymatic activities, such as glycolysis and TCA, and the Mn2+content
in wild soybean young leaves was significantly higher
than in cultivated soybean, probably because the former can maintain responses
to life processes by increasing enzymatic activities in the absence of N (He and Liu 2010). Zn2+ is an important
component of Chl synthesis, and it contributes to the ability of plants to
tolerate environmental stress factors (Koshiba 2009). In
this study, the NO3- content of the young and old leaves
of the two soybean genotypes were significantly reduced under stress
conditions, but the former decreased to a lesser extent. This corroborates
previous results in which NO3−
was transferred from old leaves to young leaves, and this may be closely related to LN tolerance
(Aoyama et al. 2014). Our research shows that wild
soybean can remain relatively stable levels of beneficial ion transport and
migration, as well as a stable nutritional balance, especially in young leaves,
which plays an important role in maintaining the growth and development of plants under LN-stress conditions.
N metabolism regulation includes N uptake, N
assimilation, ammonia assimilation, and amino acid metabolism (Wang et al. 2019). Key enzymes of N
metabolism are important regulatory factors of plant ammonia assimilation and
amino acid synthesis (Wang et al.
2012b). Here, as the LN-stress intensity increased, especially under
high-intensity stress, NR and GOT activities increased in wild soybean young
leaves, however, compared with CK, the number of young cultivated soybean
leaves is reduced. The opposite was true for the GOGAT activity. The extent of
the increase in GPT in wild soybean young leaves was greater than in cultivated
soybean. The activity levels of GS and GDH in wild soybean young leaves
decreased more than cultivated soybean young leaves. Young leaves have
increased NR and GPT activities, while old leaves have decreased NR and GPT
activities. In the two soybean genotypes, the NR activity of young leaves was
significantly higher than that of old leaves. Under LN-stress conditions,
plants may synthesize more secondary metabolites to resist the oxidation of
reactive oxygen species and alleviate the environmental stress (He et al. 2010). Additionally, as the enzymatic activity of assimilated N03−increases,
the key enzymatic activity of synthetic amino acids decreases, and most of
the NH4+ assimilated by the GS/GOGAT pathway does not
combine with the C chain produced by photosynthesis to form primary metabolite
amino acids, but is used to synthesize secondary metabolites, such as
non-protein N. The remaining C chains are used to synthesize secondary
metabolites, such as polyols and the unfavorable growth
conditions lead to polyol accumulation. Here, the NR activity increased in wild
soybean young leaves, which is consistent with results of previous studies;
however, whether NR can alleviate the LN stress remains to be determined (Li et al. 2018).
The GPT activity increased in wild soybean young leaves, but decreased in old
leaves and the GPT enzyme could regulate young leaves to control old leaves,
which affected amino acid metabolism and nutrient transport. The decrease in
the GDH activity of wild soybean young leaves was greater than that of
cultivated soybean, showing less glutamate synthesis in wild soybean and lower
amino acid metabolism and metabolic energy consumption levels in wild soybean,
which allows other sources of energy to be used for the survival and growth of
wild soybean. This may be a strategy to increase the LN
tolerance of wild soybean. The regulation of GDH plays a unique
physiological role in the processes of plant stress and aging (Cai et al. 2016). Our experiments indicated that wild soybean
could resist LN stress by regulating the activities of key enzymes involved in
amino acid synthesis, producing more non-protein N and reducing the energy
consumption of amino acid metabolism. The enzymatic activity in young leaves
was higher than in old leaves, especially under low-intensity stress, and the
young leaves could maintain higher N assimilation levels, which is beneficial
to N reuse.
C and N metabolism are tightly coupled in the
plant life-related processes (Wei et al. 2019) (Fig. 3). The reasonable regulation of C
and N metabolism by plants has significance in the integrated processes of
regulating plant growth, development, and stress (Zhu et al. 2015). The results showed that under
high-intensity stress, compared with the CK, the NR activities in the old
leaves of wild soybean and cultivated soybean
decreased significantly. This corroborated previous
results in which the cyclic electron transport was enhanced under environmental
stress conditions and the generation of NADPH was reduced, which leads to less of NR
electron donors and inhibition. The important organic acids in N
metabolism, α-OG, OAA, and pyr are derived from important %20321-331_files/image006.jpg)
Fig. 3: Comprehensive simplified model of
carbon and nitrogen metabolism
Note: This program summarizes the most important
points of interaction between carbon and nitrogen metabolism. a-KZ,
a-ketoglutaric acid; Ala, alanine; Asp, aspartic acid; Glu, glutamic acid; Gln,
glutamine; OAA, oxaloacetate; PEP, phosphoenolpyruvate; Pry, pyruvic acid
C metabolism cycles, such as
the Calvin cycle and TCA (Singh and Srivastava 1986). The synthesis of photosynthetic pigments
and the photosynthetic C metabolism cycle depend on glutamic acid and various
enzymes provided by N metabolism, and C and N metabolism also require common
reduction sources, NADPH, ATP and C skeletons, which compete with each other
(Jiang et al.
2019). C and N metabolism are highly
correlated in the young and old leaves of plants (Zhu et al. 2015). Under abiotic stress
conditions, young leaves maintain relatively stable C metabolism by maintaining
stable pigment accumulation, increasing photosynthesis processes, and energy
consumption, while C metabolism is highly reduced in old leaves (Hu et al. 2019). Additionally, young leaves
have greater abilities to maintain C and N metabolism than old leaves (Hajlaoui
et al. 2010). Improving
the metabolism of polyols in leaves and the transport strategy of polyols from
old leaves to young leaves, effectively increases the LN tolerance of wild
soybean (Li et al.
2018). Under abiotic stress conditions, the
effects on N metabolism in old leaves was greater than in young leaves (Wang et al. 2012a, b). Our study indicated
that under LN-stress conditions, wild soybean young and old leaves
maintained relatively stable balances of C and N metabolism, especially in the
young leaves. A stable C and N metabolic balance maintains the stable
production of C and N metabolites, which are transported from the old leaves to
the young leaves to support the growth and development of young leaves and
improve the LN tolerance of the leaves, thereby increasing plant resistance to
LN stress.
Conclusion
The survival and growth of plants under LN
environment depend on their physiological adjustments and metabolic changes, as
well as the interactions of C and N metabolism at the cellular level. Under
three different intensities of LN stress, the ability of wild soybean to resist
LN stress was significantly greater than cultivated soybean, and this was
closely correlated with its adaptation to a barren natural environment. This
was determined based on the following: (1) The root to shoot ratio increased by
increasing the root length; (2) The young and old leaves
of wild soybean maintained a relatively stable PN; (3) The young
and old leaves of wild soybean maintained a stable nutrient balance, and Na+,
Fe3+, Mn2+, B3+ and Zn2+ in the old
leaves were transported to the young leaves; and (4) The young leaves of wild
soybean have increased the activity of GPT and decreased the activity of GDH,
thus reduced energy consumption during amino acid metabolism.
The young and old leaves of wild soybean maintained stable dynamic balances
of C and N metabolism. This study provides a theoretical basis for the
utilization of wild soybean, the improvement of cultivated soybean, and the
study of plant resistance to abiotic stresses. The results can be used to help
improve sustainable agriculture.
Acknowledgments
Thanks to Jilin Academy of Agricultural Sciences for providing soybeans.
Funding: China National Life Science Foundation (No. 31870278); Science and
Technology Joint Innovation Project of Chinese Academy of Agricultural
Sciences.
Author Contributions
YNH, XYL, and MXL planned and designed
the research. YNH, XYL, SJG, YJH and JXG performed the experiments. YNH
analyzed the data, and XYL, MXL, SJG, and LXS wrote the manuscript. YNH, XYL,
and MXL contributed equally. All authors reviewed the manuscript.
Conflicts of Interest
The authors declare no confict of
interest.
Data Availability
Data supporting the fndings of this
study are available in the supplementary material of this article.
Ethics Approval
This article does not contain any
studies with human participants or animals. The collection materials of the
plants, complies the relevant institutional, national, and international
guidelines and legislation.
Funding Source
This study was funded by the National Natural Science Foundation of China
(No. 31870278).
References
Avice JC, P Etienne (2014). Leaf
senescence and nitrogen remobilization efficiency in oilseed rape (Brassica
napus L.). J Exp Bot 65:3813‒3824
Aoyama S, RT Huaranc, L Guglielminetti, Y Lu, Y Morita, T Sato, J Yamaguchi (2014).
Ubiquitin ligase ATL31 functions in leaf senescence in response to the balance
between atmospheric CO2 and nitrogen availability in Arabidopsis. Plant Cell Physiol 55:293‒305
Boussadia O, K Steppe, H Zgallai, SB Hadj, M Braham, R Lemeur, V Labeke (2010).
Effects of nitrogen deficiency on leaf photosynthesis, carbohydrate status and
biomass production in two olive cultivars ‘Meski’ and ‘Koroneiki’. Sci Hortic
123:336‒342
Cai HL, LD Wang, HJ Sha, HL Liu, HW Zhao (2016). Effects of nitrogen
fertilizing levels on nitrogen metabolism and grain protein content of japonica
rice in cold region under cold water stress at tillering stage. Genomics Appl Biol 35:2486‒2493
Cakmak I (2000). Possible roles of zinc in protecting plant cells form damage by
reactive oxygen species. New Phytol 146:185‒205
Cechin I, T Fumis (2004). Effect of nitrogen supply on growth and photosynthesis
of sunflower plants grown in the greenhouse. Plant Sci 166:1379‒1385
Evans J (1989). Photosynthesis and nitrogen relationships in leaves of C3
plants. Oecologia 78:9‒19
Fan SC, CS Lin, PK Hsu, SH Lin, YF Tsay (2009). The Arabidopsis nitrate transporter NRT1.7
expressed in phloem is responsible for source-to-sink remobilization of
nitrate. Plant Cell Onl
21:2750‒2761
Feller U, A Fischer (1994). Nitrogen metabolism in senescing leaves. Crit Rev Plant Sci 13:241‒241
Guo R, LX Shi, CW Yang, CR Yan, XL Zhong, Q Liu, X Xia, HR Li (2016).
Comparison of ionomic and metabolites response under alkali stress in old and
young leaves of cotton (Gossypium hirsutum L.) seedlings. Front Plant Sci 7:1758–1766
Hajlaoui H, NE Ayeb, JP
Garrec, M Denden (2010). Differential effects of salt stress on osmotic adjustment
and solutes allocation on the basis of root and leaf tissue senescence of two
silage maize (Zea mays L.) varieties. Ind Crop Prod 31:122‒130
Han ZP, SR Guo, RN Zheng
(2013). Effect of salinity on distribution of ions in mini-watermelon seedlings.
J Plant Nutr
Fert 19:908‒917
He W, X Liu (2010). Resequencing of
31 wild and cultivated soybean genomes identifies patterns of genetic diversity
and selection. Nat Genet 42:1053‒1059
Holm G (1954). Chlorophyll
mutation in barley. Acta Agric Scand 4:457‒471
Hu YJ, L Zhao, J Zhou, XL Zhong, FX Gu (2019). iTRAQ protein profile analysis
of young and old leaves of cotton (Gossypium hirsutum L.) differing in
response to alkali stress. Plant Physiol Biochem
141:370‒379
Jiang XD, MF Hua, RC Fei, LL Jiang, SB Yang, JM Guo (2019). Effects of spraying
different chemicals on dry matter accumulation and carbon and nitrogen
metabolism of rice under high temperature stress. Chin Rice 25:4‒9
Jiao Y, Z Bai, J Xu, M Zhao, Y Khan, Y Hu (2018). Metabolomics and its
physiological regulation process reveal the salt-tolerant mechanism in Glycine soja seedling roots. Plant Physiol Biochem 126:187‒196
Kong YF, XC Zhu, JF Zhang, CJ Tian (2017). Responses of drought resistance from
wild and cultivated soybeans to arbuscular mycorrhizal fungi inoculation. Soils Crops 6:25‒31
Koshiba T (2009). Boron deficiency. Plant Signal Behav 4:557‒558
Leticia G, Q Laura, L Ivan, N Ana, Catena (2019). Effects of
land use on soil organic and inorganic C and N at Cs-137 traced erosional and
depositional sites in mountain agroecosystems. Catena 181:104058
Li M, M Xu, J Xu, A Ullah, L Shi (2019). Gas Exchange and ionomic changes
in wild and cultivated soybean seedlings under salt stress. Intl J Agric Biol 22:1627–1635
Li M, J Xu, X Wang, H Fu, M Zhao, H Wang, L Shi (2018). Photosynthetic
characteristics and metabolic analyses of two soybean genotypes revealed
adaptive strategies to low-nitrogen stress. J
Plant Physiol 229:132‒141
Li M, YH Dong, QQ Cui, WD Zhang, XZ Ai, BB Liu, QM Li (2017). Effects of
water-nitrogen coupling on the metabolites and key enzyme activities of carbon
and nitrogen metabolism in cucumber leaves under doubled CO2
concentration. Plant Physiol J 53:1717‒1727
Liu XJ, MJ Wang, SB Gao, RZ Suo, LS
Wang (2017). Agronomic characteristics of hybrids from wild soybean and
cultivated soybean in inner Mongolia and their correlation with seed yield. Chin J Oil Crop Sci 39:178‒184
Liu Y, MX Li, JS Xu, XY Liu, SY Wang, LX Shi (2019). Physiological and
metabolomics analyses of young and old leaves from wild and cultivated soybean
seedlings under low nitrogen conditions. BMC
Plant Biol 19:389‒404
Loulakakis KA, KA Roubelakis-Angelakis
(1991). Plant NAD(H)-glutamate dehydrogenase consists of two subunit
polypeptides and their participation in the seven isoenzymes occurs in an
ordered ratio. Plant Physiol 97:104‒111
Mohamed AKSH, MF Qayyum, AM Abdelhadi, RA Regman, S Ali, M Rizwan
(2017). Interactive effect of salinity and silver nanoparticles on
photosynthetic and biochemical parameters of wheat. Arch Agron Soil Sci 12:1736‒1747
Mu X, Y Chen (2021). The physiological response of photosynthesis to
nitrogen deficiency. Plant Physiol Biochem
158:76‒82
Quan XY, QF Qian, ZL Ye, JB Zeng, ZG Han, GP Zhang (2016). Metabolic analysis of
two contrasting wild barley genotypes grown hydroponically reveals adaptive
strategies in response to low nitrogen stress. J Plant Physiol 206:59‒67
Rhodes D, GA Rendon, GR Stewart (1975). The control of glutamine synthetase level
in Lemnaminor L. Planta
125:201‒211
Shen W, R Guo, Y Zhao, D Liu, J Chen, N Miao, S Gao, T Zhang, L Shi (2021).
Nutrient reabsorption mechanism adapted to low phosphorus in wild and cultivated
soybean varieties. J Plant Growth Regul 2021:1–15
Shi L, S Ma, Y Fang, J Xu (2015). Crucial variations in growth and ion
homeostasis of Glycine gracilis seedlings under two types of salt stresses. J Soil Sci Plant Nutr 15:1007‒1023
Singh RP, HS Srivastava (1986). Increase in glutamate synthase (NADH) activity in
maize seedlings in response to nitrate and ammonium nitrogen. Physiol Plant 66:413‒416
Ullah A, MX Li, N Javaria, T Akash (2019). Effects of salinity on
photosynthetic traits, ion homeostasis and nitrogen metabolism in wild and
cultivated soybean. PeerJ
7:1‒27
Wang H, Z Wu, J Han, W Zheng, C Yang (2012a). Comparison of ion balance and
nitrogen metabolism in old and young leaves of Alkali-stressed rice plants. PLoS One 7:1–10
Wang H, M Zhang, R Guo, D Shi, B Liu, X Lin, C Yang (2012b). Effects of salt
stress on ion balance and nitrogen metabolism of old and young leaves in rice (Oryza
sativa L.). BMC Plant Biol 12:194‒205
Wang X, R Guo, M Li, Y Liu, M Zhao, H Fu, X Liu, S Wang, L Shi (2019).
Metabolomics reveals the drought‑tolerance mechanism in wild soybean (Glycine soja). Acta Physiol Plant 41:161‒172
Wang Y, C Zhang, Q Hao, A Sha, R Zhou, X Zhou (2013). Elucidation of
mirnas-mediated responses to low nitrogen stress by deep sequencing of two
soybean genotypes. PLoS One 8:1‒14
Wei X, LN Zhang, WZ Zhang, JP Gao, J Yi, XX Zhen, Z Li,
Y Zhao, CC Peng, C Zhao (2019). An integrated analysis of the rice transcriptome
and metabolome reveals differential regulation of carbon and nitrogen
metabolism in response to nitrogen availability. Intl J Mol Sci 20:2349‒2366
Wu LH, SH Jiang, QN Tao (1998). Method for
colorimetric determination of plant transaminase (GOT and GPT) activity and its
application. Chin J Soil Sci 29:136‒138
Wu Y, Q Li, P Dou, X Ma, D Yu, Y Luo, F Kong, J Yuan (2017). Effects of
nitrogen fertilizer on leaf chlorophyll content and enzyme activity at late
growth stages in maize cultivars with contrasting tolerance to low nitrogen. Acta Pratac Sin 26:188‒197
Yang DS, J Zhang, MX Li, LX Shi (2017). Metabolomics analysis reveals the
salt-tolerant mechanism in Glycine soja.
J Plant Growth Regul
36:460‒471
Zhao H, S Sun, L Zhang, J Yang, M Li (2020). Carbohydrate metabolism and
transport in apple roots under nitrogen deficiency. Plant Physiol Biochem 155:455‒463
Zhao ML, R Guo, MX Li, Y Liu, XX Wang, H Fu, SY Wang,
XY Liu, LX Shi (2019). Physiological characteristics and metabolomics reveal
the tolerance mechanism to low nitrogen in Glycine
soja leaves. Physiol Plant 168:1‒16
Zhu M, XL Liu, C Xu, P Ji, SY Chen,
B Liu, Y Li, X Qi (2015). Studies on photosynthetic characteristics of leaves
at different node of old and new soybean cultivars in Jilin province. J Jilin Agric Sci 40:38‒41