Introduction
A wetland has strong ecological
purification ability. In recent years, the eutrophication of wetland water is
on the rise (Tang et al. 2010; Hou et al. 2018;
Zou et al. 2018), with phosphorus being a main factor (Yu et al. 2010; Qin et al. 2013; Zhou et al.
2016; Roy 2017), which wetland plants
effectively absorb and utilize. The root system is the key bridge between
wetland plants and the survival medium; therefore, to investigate energy
circulation (Akhtar et al.
2016; You et al. 2013; Zhang et al. 2018a; b), it is essential to study root system exudates.
Studies have shown that organic acids secreted by roots
can regulate rhizosphere micronutrients and change the structural and
physicochemical properties of root microbes in a plant’s growth environment.
Organic acids can also regulate plant resistance to adverse environments and
relieve hypoxia; therefore, studying them aids in the understanding of the
mechanism of plant resistance (Hinsinger
2001; Kuang et al. 2003;
Rellan-Alvarez et al. 2010; Zhao et al. 2016). The effects of phosphorus stress on the secretion of organic acids by
plant roots have been investigated. For example, rice roots excrete more
organic acids under low phosphorus stress ( Li et al. 2005; Deng et al.
2006; Wang et al. 2014); while some plants induce phosphorus release from
soil by changing root morphology and increasing secretion of organic acids in a
phosphorus-deficient environment (Zhou et al. 2011; Ma et al.
2017). Low molecular weight organic acids in
root exudates of wheat and broad bean could improve the bioavailability of
phosphorus. In addition, Deng et al. (2006) studied the effect of phosphorus concentration on root exudates of
Pinus yunnanensis seedlings, demonstrating the changes in organic acids
in the exudates of P. yunnanensis seedlings. The root exudates of
wetland plants primarily consisted of small molecular organic acids and
aromatic proteins (Lu et al.
2009; Huang et al. 2014). Previous
studies have focused on the effects of low phosphorus stress on cash crops and
trees, as well as root exudation and rhizosphere effects of wetland plants.
Although the effects of plant root exudates and phosphorus stress on plant
roots have been investigated, there are few studies regarding the response
mechanism in root exudates of submerged plants to phosphorus stress as well as
the effect of phosphorus stress on organic acid secretion. Therefore, this
study aimed to understand the stress response of root exudates to pollution and
to explain the mechanism of phytoremediation.
Hydrilla verticillata is a
submerged herb. Some studies have shown that Hydrilla can transform
phosphorus from wetland sediment to be utilized, and it can purify the water
after eutrophication (Zhao et al.
2008; Li et al. 2016).
Materials and Methods
Water culture and solution ratio
The Hoagland nutrient solution was
used in the laboratory of Southwest Forestry University at the beginning of
March to culture Hydrilla seedlings at 20–30°C. Phosphorus stress was
induced at the beginning of April, and was measured at 7, 14, 21, and 28 days.
Eighty plastic barrels were used as hydroponic equipment, where the Hoagland
nutrient solution was added. Healthy Hydrilla, 160, with insignificant
differences in seedling age were selected and 2 seedlings were cultured in each
barrel. In the barrels containing the plants, a 300 mm rubber disc microporous
aerator was installed to prevent the plant root system from rotting by ensuring
a sufficient amount of aeration. To avoid the algal blooming in the barrel,
aluminum foil was used for shading (Horchani et al. 2008). The culture medium was changed once a week. After one
month of culture, Hydrilla plants with good growth and similar height
were selected and transplanted into the experimental device. Monopotassium
phosphate (KH2PO4) was used as the phosphorus source,
with a phosphorus concentration gradient of 0, 0.2, 1, 5, 10, and 20 mg•L-1.
There were 72 plastic barrels (4 groups of experiments, 3 replicates in each
group), and 2 plants were cultured in each barrel.
Hoagland nutrient solution contained: potassium sulfate (K2SO4):
0.75×10-3 mol•L-1, magnesium sulfate (MgSO4):
0.75×10-3 mol•L-1, potassium chloride (KCl): 1×10-3 mol•L-1,
calcium nitrate (Ca(NO3)2): 2.0×10-3 mol•L-1,
Boric acid (H3BO3): 1×10-5 mol•L-1,
copper(II) sulfate (CuSO4): 1×10-7 mol•L-1,
manganese(II) sulfate (MnSO4): 1×10-6 mol•L-1,
zinc sulfate (ZnSO4): 1×10-6 mol•L-1, ammonium
heptamolybdate ((NH4)6Mo7O24): 5×10-6 mol•L-1,
Fe-EDTA (C10H12FeN2O8): 1×10-4 mol•L-1. This stock
was 4× diluted for the hydroponic culture (Chen, 2009).
Collection and isolation of root
exudates
The
entire root system of the plant cultured under phosphorus stress was washed
with deionized water for 7, 14, 21, and 28 days, and root exudates were
collected, (collection solution ratio: 5 μmol•L-1
H3BO3, 600 μmol•L-1 calcium
chloride (CaCl2), 100 μmol•L-1 KCl, and 200 μmol•L-1
magnesium chloride (MgCl2), pH 5.6). After repeated washing for
three times, the root system was covered with a black plastic bag, and
transferred to a beaker containing 50 mL root exudates. The root exudates were
collected under natural light for 4 h (9:00–13:00). The root lotion was
obtained by transplanting the root in solution containing 1 L of 0.5 mmol L-1
CaCl2 for 4 h (13:00–17:00) (Tian
et al. 2003; Zhang et al. 2007). The extraction
solution was obtained using dichloromethane (CH2Cl2)
extraction and root lotion for three times (40 mL/times). Finally, 200 mL
extraction solution was collected at 38℃, dried with anhydrous (Na2SO4),
and then concentrated to dry reserve by vacuum rotary evaporator (Wei et al. 2016).
Determination of root exudates
The solution was extracted with a
syringe after a full shake of CH2Cl2, which was added to
the rotating evaporation at 1.2 knots for 0.5 mL, after passing through a 0.45 μm needle filtration membrane.
Meanwhile, the membrane was filtered using a 0.45 μm needle and then put into a small brown bottle for further
GC-MS analysis. The organic acids in root exudates were determined by gas
chromatography/mass spectrometry (Agilent 7890B). The chromatographic
conditions were as follows: the capillary column was HP-5 ms column (30 m × 250
μm × 0.25 μm) (Huiyong et al.
2013; Liu et al. 2017), the injection
port temperature was 260°C, the carrier gas was helium (He) (purity of
99.999%), the flow rate was 1 mL min-1 with injection volume of 1 μL,
the flow valve was opened after 1 min, the column temperature was programmed,
starting at 50°C, with flow rate of 2 min; 20°C per minute, programmed to 150°C,
and 5°C per minute, programmed to 220°C, then, 6°C per minute, programmed to 250°C,
for 15 min.
As for the mass spectrum conditions (Liu et al. 2017), electron bombardment source (EI) ionization energy
was 70eV, ion source temperature was 200°C while the interface temperature was 280°C
and quadrupole temperature was 150°C, solution delay time was for 3.75 min,
scanning mode (SCAN) with a scanning range of M/Z 33-453, and standard tuning.
Data analysis
The organic acids in Hydrilla root exudates were
identified by artificial analysis of total ion flow map and referencing it with
the standard map of NIST08 mass spectrometry database. The determination of
root exudates was carried out by computer search. The relative concentration of
substances was calculated according to the peak area (%) of the components
which is detected in the chromatogram.
Data processing and statistical methods
In this study, excel WPS2016 and SPSS21
software were used for data processing and statistical analysis. The LSD method
was used for multiple comparisons. The significant level, α, was 0.05,
while the extremely significant level α was 0.01, and the scanning map was
drawn by origin8.5.
Results
The organic acids and relative
contents in root exudates of H. verticillata were determined under
different phosphorus concentration gradient stress by artificially analyzing
the GC-MS scanning map. Six different phosphorus stress conditions were used
and the results were referenced with the standard map of NIST08 mass spectrometry
database. The organic acids secreted in response to six concentrations of
phosphorus stress and four time periods were analyzed, and were classified into
the total organic acids.
GC-MS scanning pattern of root
exudates from H. verticillata
In this paper, only the scanning of
four different time periods, at phosphorus concentration of 20 mg•L-1
is listed. The results of scanning chart 1 show that the scanning spectra are
different at different time periods, and the number of characteristic peaks as
well as the areas of high and low peaks are not consistent. Each characteristic
peak represents a compound, and thus scanning spectra indicates that the
secretion of root exudates under phosphorus stress differs depending on the
time periods.
Differences in organic acids
secretion in the roots of H. verticillata at different phosphorus
concentrations and stress periods
Changes in organic acid secretion under
different phosphorus concentrations during the same stress period: Table 1 shows that when phosphorus stress time was
at 7 days, the secretion of carbonic acid, phthalic acid, phenyldicarboxylic
acid, benzoic acid and total organic acid was the lowest (phosphorus
concentration is 0 mg/L), with relative content of: 0.11, 3.09, 0.51, 0.01 and
7.85%, respectively. Under a concentration of 20 mg/L phosphorus, the exudation
of stone carbonate and phenyldicarboxylic acid was the highest. No significant
differences were observed in secretion at phosphorus concentration of 10 mg/L
and 20 mg/L. The secretion of benzodicarboxylic acid initially decreased then
increased. There was no significant difference in secretion between 0 mg/L and
0.2 mg/L phosphorus concentration. The highest secretion of benzoic acid and
phthalic acid was observed when phosphorus concentration was 10 mg/L, and then
decrease as phosphorus concentration increased. There was no significant
difference in benzoic acid secretion between 1, 10 and 20 mg/L phosphorus
concentration. The maximum secretion of total organic acids was observed when
the concentration of phosphorus was 1 mg/L. The lowest secretion of sulfurous
acid and carboxylic acid was observed when phosphorus concentration was 10
mg/L. The secretion at 0–10 mg/L phosphorus concentration decreased significantly
in the range concentration of sulfite. The secretion at 20 mg/L phosphorus
concentration was significantly higher than that of the 10 mg/L. There was no
significant difference in carboxylic acid between 5, 10 and 20 mg/L phosphorus
concentration, in which the minimum secretion of carboxylic acid was observed
at 0.2 mg/L phosphorus concentration. Oxalic acid secretion gradually decreased
as the phosphorus concentration increased. The maximum amount of sulfonic acid
was observed when concentration of phosphorus was 0 mg/L while the minimum
amount was observed at 5 mg/L.
When phosphorus stress lasted for
14 days, the highest amount of excretion was obtained from phthalic acid at
36.45%, phenyldicarboxylic acid at 4.20%, sulfonic acid at 0.59%, and total organic
acid at 65.88% under phosphorus concentration of 1 mg/L. The contents of
phthalic acid and phthalic acid was the least when phosphorus concentration was
0 mg/L. There was no significant difference between the two acids in the
relative content at 10 and 20 mg/L phosphorus concentration; additionally, in
the range of 1–10 mg/L phosphorus concentration, the content of the two acids
decreased significantly. The secretion of sulfonic acid and total organic acid
of 1 mg/L was significantly higher than that of 0 mg/L. The lowest secretion of
sulfonic acid and total organic acid was 0.23 and 16.12% under 5 mg/L
phosphorus concentration respectively, in the 1–20 mg/L region, the secretion
decreased significantly under 20 mg/L phosphorus concentration. The secretion
of carbonates, carboxylic acids and benzoic acid was the lowest when phosphorus
concentration was 0 mg/L, and the order of relative content was: 0.32, 0.41 and
0.22%, respectively. While the concentration of carboxylic acid and benzoic
acid at 1 mg/L phosphorus was higher than that of 0 mg/L, there was no
significant difference in the secretion of carboxylic acid between 0.2, 10, and
20 mg/L. There was no significant difference in the content of benzoic acid
under phosphorus concentrations between 1, 10, 5, and 20 mg/L. The maximum
secretion of carbonic acid was 17.43%, when the concentration of phosphorus was
0.2 mg/L, and the amount of excretion between 0 and 20 mg/L decreased
significantly. The maximum amount of sulfite excretion was 1.06% in the 20 mg/L
phosphorus concentration treatment, and at least 0.26% in 0.2 mg/L phosphorus
concentration treatment, while the amount of secretion between 1 and 20 mg/L
increased gradually. The maximum excretion of oxalic acid, 0.59%, was observed
in the 0.2 mg/L phosphorus concentration treatment, and at least 0.21% in the 5
mg/L phosphorus concentration treatment. There was no significant difference in
the secretion of sulfurous acid between 5, 1, and 20 mg/L phosphorus
concentration treatments.
Table 1:
Comparison of relative content of organic acids in root exudates of H.
Verticillata under different phosphorus concentration and time treatment
(%)
|
Organic acid |
Concentration/(mg/L) |
Duress time/(day) |
|||
|
7 |
14 |
21 |
28 |
||
|
Sulfurous acid |
0 |
1.15
± 0.03 a-A |
0.44
± 0.04 c-C |
0.49
± 0.06 b-BC |
0.56
± 0.04 d-B |
|
0.2 |
0.94
± 0.03 b-B |
0.26
± 0.06 d-D |
0.46
± 0.04 b-C |
1.11
± 0.04 b-A |
|
|
1 |
0.59
± 0.05 d-B |
0.44
± 0.03 c-B |
0.40
± 0.08 b-B |
1.37±
0.14 a-A |
|
|
5 |
0.47
± 0.04 e-B |
0.87
± 0.06 b-B |
0.46
± 0.05 b-C |
1.06
± 0.10 b-A |
|
|
10 |
0.37
± 0.07 f-B |
0.94
± 0.07 b-A |
0.90
± 0.04 a-A |
0.60
± 0.09 cd-B |
|
|
20 |
0.80
± 0.06 c-BC |
1.06
± 0.05 a-A |
0.88
± 0.05 a-B |
0.74
± 0.08 c-C |
|
|
Phenol |
0 |
0.11
± 0.02 e-C |
0.32
± 0.02 f-B |
0.87
± 0.05 e-A |
0.34
± 0.04 d-B |
|
0.2 |
1.03
± 0.15 d-C |
17.43
± 0.08 a-A |
9.68
± 0.08 c-B |
0.53
± 0.06 c-D |
|
|
1 |
6.25
± 0.06 c-C |
12.65
± 0.05 c-A |
9.51
± 0.03 d-B |
0.62
± 0.05 b-D |
|
|
5 |
6.28
± 0.06 bc-C |
16.64
± 0.07 b-A |
10.14
± 0.07 b-B |
0.79
± 0.03 a-D |
|
|
10 |
6.49
± 0.04 a-B |
1.69
± 0.11 d-C |
9.60
± 0.06 cd-A |
0.33
± 0.02 d-D |
|
|
20 |
6.53
± 0.05 a-B |
1.38
± 0.10 e-C |
10.26
± 0.06 a-A |
0.20
± 0.01 e-D |
|
|
Carboxylic Acid |
0 |
0.54
± 0.06 c-A |
0.41
± 0.03 c-B |
0.31
± 0.04 c-C |
0.56
± 0.02 f-A |
|
0.2 |
1.26
± 0.05 a-B |
0.77
± 0.06 b-C |
0.28
± 0.04 c-D |
2.01
± 0.03 a-A |
|
|
1 |
0.72
± 0.10 b-C |
0.86
± 0.07 ab-B |
0.12
± 0.02 d-D |
1.95
± 0.05 b-A |
|
|
5 |
0.38
± 0.04 d-C |
0.92
± 0.05 a-B |
0.45
± 0.05 b-C |
1.27
± 0.04 d-A |
|
|
10 |
0.34
± 0.02 d-C |
0.76
± 0.09 b-B |
1.13
± 0.12 a-A |
0.94
± 0.03 e-D |
|
|
20 |
0.39
± 0.04 d-D |
0.71
± 0.03 b-C |
1.06
± 0.10 a-B |
1.76
± 0.02 c-A |
|
|
Phthalic acid |
0 |
3.09
± 0.02 c-B |
3.96
± 0.07 d-A |
3.98
± 0.17 b-A |
0.36
± 0.01 c-B |
|
0.2 |
3.52
± 0.11 c-B |
27.67
± 1.04 b-A |
27.10
± 0.17 a-A |
0.97
± 0.05 a-B |
|
|
1 |
28.20
± 1.80 b-B |
36.45
± 1.03 a-A |
26.23
± 1.49 a-B |
0.43
± 0.02 bc-C |
|
|
5 |
29.73
± 1.54 ab-A |
9.92
± 0.26 c-C |
27.01
± 1.05 a-B |
0.49
± 0.04 b-D |
|
|
10 |
30.84
± 1.51 a-A |
6.90
± 1.05 d-B |
3.38
± 0.22 b-C |
0.34
± 0.05 c-D |
|
|
20 |
29.90
± 1.57 ab-A |
7.84
± 0.96 d-B |
0.51
± 0.03 c-C |
0.37
± 0.03 c-C |
|
|
Oxalic acid |
0 |
0.70
± 0.09 a-A |
0.41
± 0.08 b-B |
0.55
± 0.11 a-B |
0.06
± 0.01 d-C |
|
0.2 |
0.57
± 0.09 b-A |
0.59
± 0.06 a-A |
0.52
± 0.04 b-A |
0.43
± 0.04 b-B |
|
|
1 |
0.49
± 0.05 bc-AB |
0.33
± 0.05 bc-B |
0.39
± 0.10 c-B |
0.56
± 0.03 a-A |
|
|
5 |
0.39
± 0.03 c-B |
0.21
± 0.04 c-C |
0.25
± 0.03 d-C |
0.53
± 0.03 a-A |
|
|
10 |
0.30
± 0.04 cd-B |
0.44
± 0.05 b-A |
0.50
± 0.05 bc-A |
0.23
± 0.04 c-B |
|
|
20 |
0.28
± 0.04 d-B |
0.27
± 0.04 c-B |
0.67
± 0.06 a-A |
0.18
± 0.03 c-C |
|
|
Benzene dicarboxylic acid |
0 |
0.51
± 0.04 e-B |
0.60
± 0.09 e-B |
0.78
± 0.06 f-A |
0.11
± 0.02 b-C |
|
0.2 |
0.54
± 0.05 e-C |
3.19
± 0.08 b-A |
2.93
± 0.05 e-B |
0.23
± 0.03 a-D |
|
|
1 |
3.62
± 0.04 b-B |
4.20
± 0.07 a-A |
3.07
± 0.02 d-C |
0.09
± 0.02 b-D |
|
|
5 |
3.48
± 0.09 c-A |
1.19
± 0.02 c-C |
3.32
± 0.04 c-B |
0.08
± 0.01 bc-D |
|
|
10 |
3.26
± 0.09 d-B |
0.76
± 0.10 d-C |
3.45
± 0.09 b-A |
0.05
± 0.01 c-D |
|
|
20 |
3.74
± 0.05 a-A |
0.88
± 0.07 d-B |
3.69
± 0.12 a-A |
0.03
± 0.00 c-C |
|
|
Benzoic Acid |
0 |
0.01
± 0.00 d-D |
0.22
± 0.02 d-C |
0.78
± 0.05 d-B |
0.89
± 0.09 d-A |
|
0.2 |
0.22
± 0.01 c-D |
1.12
± 0.11 b-B |
1.78
± 0.05 b-A |
0.99
± 0.05 d-C |
|
|
1 |
0.97
± 0.10 a-C |
1.28
± 0.06 a-B |
1.96
± 0.06 a-A |
1.26
± 0.04 c-B |
|
|
5 |
0.71
± 0.04 b-D |
0.77
± 0.06 c-C |
1.47
± 0.05 c-B |
2.11
± 0.09 b-A |
|
|
10 |
1.04
± 0.04 a-C |
1.37
± 0.08 a-B |
0.28
± 0.06 f-D |
3.85
± 0.22 a-A |
|
|
20 |
1.00
± 0.18 a-A |
0.88
± 0.04 c-A |
0.49
± 0.05 e-B |
0.23
± 0.04 e-C |
|
|
Sulfonic acid |
0 |
0.58
± 0.02 a-A |
0.32
± 0.03 b-B |
0.09
± 0.03 f-D |
0.23
± 0.05 e-C |
|
0.2 |
0.16
± 0.02 d-D |
0.52
± 0.04 a-B |
0.31
± 0.03 e-C |
1.84
± 0.05 a-A |
|
|
1 |
0.18
± 0.02 cd-C |
0.59
± 0.04 a-B |
0.59
± 0.05 d-B |
1.57
± 0.06 b-A |
|
|
5 |
0.22
± 0.03 c-B |
0.23
± 0.04 c-B |
0.68
± 0.04 c-A |
0.30
± 0.06 e-B |
|
|
10 |
0.28
± 0.03 b-D |
0.53
± 0.05 a-C |
1.12
± 0.05 b-B |
1.27
± 0.08 c-A |
|
|
20 |
0.31
± 0.03 b-B |
0.31
± 0.03 b-B |
1.34
± 0.06 a-A |
0.92
± 0.03 d-C |
|
|
Total organic acid |
0 |
7.85
± 0.21 c-A |
6.68
± 0.19 f-B |
7.85
± 0.37 d-A |
3.11
± 0.05 d-C |
|
0.2 |
8.54
± 0.20 c-C |
55.13
± 1.45 b-A |
49.60
± 0.10 a-B |
9.86
± 0.28 b-C |
|
|
1 |
49.48
± 1.80 a-B |
65.88
± 1.17 a-A |
48.64
± 1.41 a-B |
9.95
± 0.30 b-C |
|
|
5 |
46.29
± 1.43 b-B |
32.95
± 0.47 c-C |
49.55
± 1.06 a-A |
8.29
± 0.15 c-D |
|
|
10 |
47.86
± 1.62 ab-A |
18.89
± 1.76 d-C |
22.75
± 0.46 b-B |
11.90
± 0.23 a-D |
|
|
20 |
47.62
± 1.68 ab-A |
16.12
± 1.14 e-C |
19.90
± 0.27 c-B |
9.63
± 0.05 b-D |
|
Note: the figures in the table were all
repeated mean ±standard errors in three groups. The lowercase letters after the
numbers in the table indicate the differences (longitudinally) in secretions
between different concentrations of phosphorus in the same organic acids at the
same time of stress, The uppercase letters represent the differences
(horizontal) between different stress time treatments of the same organic acid
at the same phosphorus concentration. In the same column (or row), the same
letter means that the difference is not significant (p > 0.05), while the
letter difference means the difference is significant
When phosphorus stress duration was 21 days, the highest secretion of
phenyldicarboxylic acid, sulfonic acid and oxalic acid appeared at the stress
of phosphorus concentration of 20 mg/L, and the relative content was as follows:
3.69, 1.34 and 0.67%, respectively. Both phthalic acid and sulfonic acid as
well as oxalic acid excretions increased significantly with the increase in
phosphorus concentration, with no significant difference between 0.2 and 10
mg/L phosphorus concentration in the secretion of oxalic acid. The minimum
amount of excretion was 0.25% at phosphorus concentration 5 mg/L. The maximum
exudation of carbonic acid, phthalic acid and total organic acid were all at
the concentration of 0.2 mg/L, while the minimum secretion of carbonated stone
and total organic acid was both observed in the 0 mg/L phosphorus
concentration, and the minimum secretion of phthalic acid was 0.32% in the 0
mg/L phosphorus concentration. There was no significant difference between
total organic acid and phthalic acid under the phosphorus concentrations of
0.2, 1, and 5 mg/L, but the secretion under 0.2 mg/L was significantly higher
than that under 0 mg/L. The secretion of carbonic acid increased significantly
under 0–5 mg/L; the maximum amount of sulfurous acid and carboxylic acid
secreted was at 10 mg/L, and the least amount of acid excretion observed in the
1 mg/L phosphorus treatment. There was no significant difference between the
two acids at the concentration of 10 and 20 mg/L, and 0 and 0.2 mg/L. The
maximum secretion of benzoic acid was 1.96% where phosphorus concentration was
1 mg/L, and the minimum secretion of benzoic acid was 0.28% when phosphorus
concentration was 10 mg/L. The secretion of benzoic acid increased
significantly in 0–1 mg/L. There was a significant decrease in secretion
between 5 and 20 mg/L.
The maximum secretion of carboxylic acid, phthalic acid, benzoic acid, and
sulfonic acid appeared at 28 days of phosphorus stress, when phosphorus concentration
was 0.2 mg/L, and the relative content of carboxylic acid, phthalic acid,
benzoic acid, and sulfonic acid in turn was 2.01, 0.97, 0.23 and 1.84%,
respectively. The secretion of above four acids in 0.2 mg/L was significantly
higher than that in 0 mg/L. In the range of 0.2–20 mg/L concentration, the
excretion of phthalic acid decreased with the increase in phosphorus
concentration. There was no significant difference in secretion between 0, 1,
10, and 20 mg/L, and no significant difference in the relative content of
phthalic acid and benzene dicarbonic acid in the 1 and 5 mg/L phosphorus
concentration treatments. The secretion of sulfonic acid in response to 0.2–5
mg/L phosphorus concentration was significantly decreased; the content of
benzoic acid and total organic acid was the highest in the 10 mg/L phosphorus
concentration treatment. The content of benzoic acid in 20 mg/L was
significantly larger than that in 10 mg/L, and the amount of benzoic acid in
response to 0–10 mg/L increased with the increase in phosphorus concentration.
However, there was no significant difference in secretion between 0 and 0.2
mg/L. The secretion of sulfurous acid and oxalic acid was the highest when
phosphorus concentration was 1 mg/L, and it was the lowest at the 0 mg/L. The
secretion of the two acids in the 0–1 mg/L region was significantly increased,
but there was no significant difference in secretion at the phosphorus
concentrations of 10 and 20 mg/L. The secretion of sulfurous acid in the 1–10
mg/L region decreased significantly, so did the oxalic acid in the 1–20 mg/L
region. As phosphorus concentration increased, the secretion gradually
decreased. The secretion of carbonaceous acid increased significantly in the 0–5
mg/L concentration and decreased significantly in the 5–20 mg/L concentration.
Changes in organic acid secretion in
different time periods under the same phosphorus concentration stress
Table 1 shows that when phosphorus
concentration was 0 mg/L, the maximum secretion of sulfurous acid, oxalic acid,
sulfonic acid and total organic acid was observed at 7 days, while the minimum
secretion of oxalic acid and total organic acid was at 28 days. The excretion
of oxalic acid decreased gradually with the increase in culture time, but there
was no significant difference in the secretion of total organic acid between 7
and 21 days. The minimum secretion of sulfite was 0.44% after 14 days of
culture, and there was no significant difference between 14, 21, and 28 days,
but the amount of secretion gradually decreased between 7 and 28 days. The
secretion of sulfonic acid and phthalic acid increased significantly between 7
and 21 days, and the secretion of 28 days was significantly lower than that of
21 days. While the maximum secretion of carboxylic acid and benzoic acid was
observed at 28 days, the secretion of carboxylic acid decreased significantly
from 7 to 21 days, and the secretion of benzoic acid increased with the
increase in culture time. The maximum secretion of phthalic acid and benzene
dicarbonic acid was 21 days, and the secretion of phthalic acid increased
significantly between 7 and 21 days, but there was no significant difference
between 14 and 21 days of culture period. The minimum secretion of benzene
dicarbonic acid was 0.11% at 28 days, and there was no significant difference
between 7 and 14 days.
When phosphorus concentration was 0.2 mg/L, the secretion of sulfurous
acid, carboxylic acid and sulfonic acid was the highest at 28 days, the
relative content was 1.11, 2.01 and 1.84%, respectively. The secretion of
sulfurous acid and carboxylic acid decreased significantly in the range of 7–21
days. The secretion at 28 days was significantly larger than that of 21 days,
while the minimum secretion of sulfonic acid was 0.16% at 7 days, the secretion
at 28 days was significantly higher than that of the other three periods. The
maximum exudation of carbonate, phthalic acid, total organic acid, and benzene
dicarbonic acid were at 14 days, the secretion of all four acids in 14 days was
significantly greater than that at 7 days. The secretion of phthalic acid and
benzene dicarbonic acid decreased from 21 to 28 days, and the secretion of
lithic acid at 28 days was significantly lower than that of the other three
periods. The secretion of total organic acid decreased significantly from 14 to
21 days; the secretion of oxalic acid decreased gradually with the increase in
culture time, and there was no significant difference between 7, 14, and 21
days. The secretion of benzoic acid increased significantly from 7 to 21 days,
and the relative content of benzoic acid at 28 days was significantly lower
than that of 21 days.
When phosphorus concentration was 1 mg/L, the secretion of sulfurous acid,
carboxylic acid, oxalic acid, and sulfonic acid was the highest when cultured
for 28 days and the relative content is shown in Table 1, Fig. 1. The secretion
of sulfurous acid in 28 days was significantly higher than that in the other
three periods. There was no significant difference in the relative content of
sulfonic acid between 7, 14, and 21 days. The secretion of sulfonic acid
increased significantly with the increase in culture time, and the secretion of
carboxylic acid at 28 days was significantly higher than that in other periods;
there was no significant difference in the secretion of oxalic acid at 7, 14,
and 21 days. The total organic acid, phthalic acid, total organic acid, and
phenyldicarboxylic acid secretions were the highest at 14 days and the lowest
at 28 days. The secretion of three kinds of acids and total organic acids in 14
days were significantly higher than those in 7 days, and the secretion at 14 to
28 days decreased significantly. There was no significant difference in the
secretion of phthalic acid between 7 and 21 days. The secretion of benzoic acid
increased significantly in 7–21 days and decreased significantly in 28 days.
%201033-1043_files/image002.png)
Fig. 1:
Scanning map of root exudates of H. Verticillata at different times of
20 mg/L phosphorus concentration
The specific relative content of each acid when phosphorus concentration
was 5 mg/L is shown in Table 1. The secretion of sulfurous acid, carboxylic
acid, oxalic acid, and benzoic acid appears at most in 28 days, the relative
content in turn is 1.06, 1.27, 0.53 and 2.11%, respectively. Oxalic acid,
sulfurous acid, and carboxylic acid showed the same regularity of decreasing
and then increasing with the change of culture time. The secretion of oxalic
acid at 28 days was significantly higher than that at other three periods, and
there was no significant difference between 14 and 21 days of oxalic acid
secretion. The secretion of benzoic acid increased with the increase in culture
time. The amount of sulfonic acid and total organic acid was the highest at 21 days,
but the secretion of sulfonic acid on days 7, 14, and 21 had no significant
difference, and the total organic acid content at day 28 was significantly
lower than that at the other three periods. The maximum secretion of phthalic
acid and benzene dicarbonic acid was observed at 7 days and least at 28 days.
With the increase in culture time, the secretion of stony carbonic acid
decreased first and then decreased, and the secretion of carbonate at 14 days
was significantly higher than that at 7 days, and the secretion from 14 to 28
days decreased significantly.
When phosphorus concentration was 10 mg/L, the maximum secretion of
carbonated acid, carboxylic acid, oxalic acid, and phenyldicarboxylic acid was
at 21 days, the minimum secretion of stone carbonate, oxalic acid, and
phenyldicarboxylic acid was at 21 days, and the minimum secretion of carboxylic
acid was at 7 days. The secretion of carboxylic acid and oxalic acid increased
significantly from 7 to 21 days, and the secretion at 28 days was significantly
lower than that at 21 days. Secretion of phthalene dicarboxylic acid and
carbonic acid changed with the duration of culture and then increased and then
decreased significantly. The specific relative content is shown in Table 1. The
highest secretion of benzoic acid and sulfonic acid was observed at 28 days,
the minimum secretion of benzoic acid was at 21 days, and the amount of
sulfonic acid increased significantly with the increase in culture time. Both
phthalic acid and total organic acid showed a significant decrease with the
increase in culture time.
When phosphorus concentration was 20 mg/L, the secretion of phthalic acid,
phenyldicarboxylic acid, benzoic acid, and total organic acid was the highest
at 7 days and the lowest at 28 days. The relative content is shown in Table 1.
The secretion of phthalic acid and total organic acid increased significantly
with the increase in culture time, and the secretion of benzoic acid increased
gradually with the increase in culture time. There was no significant difference
in the secretion of benzoic acid at 7 and 14 days. There was no significant
difference in the secretion of benzene dicarbonic acid between 7 and 21 days.
The secretion of stony carbonate and oxalic acid increased significantly from 7 to 21 days being the highest at 21
days and lowest at 28 days. The secretion of sulfurous acid initially increased
followed by a decrease, and the secretion volume decreased significantly from
14 to 21 days. Carboxylic acid secretion increased significantly with the increase
in stress duration.
%201033-1043_files/image004.png)
Fig. 2:
Correlation analysis between phosphorus concentration and organic acid at 7
days of stress duration
Correlation analysis between phosphorus
concentration and relative content of organic acids in roots
By correlation analysis, we found a
significant positive correlation between the concentrations of carbonic acid,
phthalic acid, phenyldicarboxylic acid and benzoic acid in root exudates of H.
verticillata cultured for 7 days (Fig. 2); (r = 0.610, P = 0.007; r =
0.611, P = 0.007; r = 0.607, P = 0.008; r = 0.633, P = 0.005). The results
showed that with the increase in phosphorus stress concentration, the secretion
of carbonic acid, phthalic acid, phenyldicarboxylic acid and benzoic acid also
increased, and the total organic acid had a significant positive correlation
with phosphorus stress concentration (r = 0.573, P = 0.013), and significant
negative correlation between oxalic acid and phosphorus stress concentration (r
= -0.787, P = 0.000). The relative content of oxalic acid decreased as the
phosphorus concentration increased, additionally, there is a significant
negative correlation between carboxylic acid and phosphorus stress
concentration, (r = -0.564, P = 0.015), There was no significant correlation
between sulfurous acid and sulfonic acid contents and phosphorus stress
concentration at 7 days of culture (P > 0.05). When the stress
duration was 14 days (Fig. 3), there was a very significant positive
correlation between the concentration of sulfite and phosphorus stress
(r=0.865, P=0.000). There was a significant negative correlation between
carbonic acid and phenyldicarboxylic acid and phosphorus stress concentration
(r = -0.500, P = 0.035; r = -0.493, P = 0.037), that is, with the increase in
phosphorus concentration, the exudation of carbonate and phenyldicarboxylic
acid in root exudates decreased gradually after 14 days of culture. The
relative contents of carboxylic acid, phthalic acid, oxalic acid, benzoic acid, sulfonic acid, and total
organic acid had no significant correlation with phosphorus concentration after
14 days of culture (P > 0.05). When the stress duration was 21 days
(Fig. 4), there was a very significant positive correlation between sulfurous
acid, carboxylic acid and sulfonic acid concentration and phosphorus stress
concentration (r = 0.847, P = 0.000; r = 0.864, P = 0.000; r = 0.919, P =
0.000). There was a significant positive correlation between benzene dicarbonic
acid and phosphorus stress concentration (r = 0.589, P = 0.010), and very
significant negative correlation between phthalic acid, benzoic acid and
phosphorus stress concentration (r = -0.616, P = 0.006; r = -0.669, P = 0.002).
There was no significant correlation between the amount of calcium carbonate,
oxalic acid and total organic acid in phosphate stress culture for 21 days (P
> 0.05). When the stress duration was 28 days (Fig. 5), there was a
significant negative correlation between carbonation and phosphorus stress
concentration (r = -0.575, P = 0.013). There was a significant negative
correlation between benzene dicarbonic acid and phosphorus stress concentration
(r = -0.678, P = 0.002), with the increase in phosphorus concentration, the amount
of carbonate and benzene dicarbonic acid in root exudates at 28 days decreased
gradually. Sulfurous acid, carboxylic acid, phthalic acid, oxalic acid, benzoic
acid, sulfonic acid, and total organic acid had no significant correlation with
phosphorus concentration at 28 days of phosphorus stress (P > 0.05).
%201033-1043_files/image006.png)
Fig. 3:
Correlation analysis between phosphorus concentration and organic acid at 14
days of stress duration
%201033-1043_files/image008.png)
Fig. 4:
Correlation analysis between phosphorus concentration and organic acid at 21
days of stress duration
%201033-1043_files/image010.png)
Fig. 5:
Correlation analysis between phosphorus concentration and organic acid at 28
days of stress duration
Discussion
Root system is an
important part of energy exchange between plants and the surrounding
environment. Plants adjust root exudate types and contents to adapt to
environmental changes and play a major role in regulating the supply of
micronutrient elements in the rhizosphere. The diversity of root exudates is
the result of adaptation of different species to their living media (Tian et al. 2000; Qin et al. 2011; Chen
et al. 2017). Root exudates can
change the environment of plant roots by adjusting the secretion of organic
acids, activate insoluble phosphorus in nutriental elements, and increase the
absorption of nutrients in plants (Duan 2003; Zhang 2009). In the
wetland’s ecosystem where eutrophication is becoming a serious concern, the
study on the effects of phosphorus stress on root exudates of wetland plants
can clarify the rhizosphere measures of wetland management.
With the increase in culture time and phosphorus stress concentration, the
amount of organic acids in H. verticillata root exudates varied greatly
at different concentration levels and culture periods. After 7 days of culture,
organic acids in H. verticillata roots exudates, such as carbonic acid,
phthalic acid, benzoic acid, and total organic acid, increased with the
increase in phosphorus concentration. At 14 days, only sulfite secretion
increased with phosphorus concentration, while at 21 days, the secretion of
sulfurous acid, carboxylic acid, phenyldicarboxylic acid, and sulfonic acid
increased with the increase in phosphorus concentration, and no increase was
observed at day 28. The reason for this phenomenon may be that at 7 days, the
plant was exposed to phosphorus stress environment, the root system was sensitive,
and the rhizosphere environment was regulated by increasing the exudation of
organic acid. H. verticillata is a wetland plant, with a fast growth
rate with culture time. As H. verticillata slowly approaches the aging
stage, where the root secretion capacity is weakened, it may lead to the
reduction in acid secretion. The reasons for the decrease in specific
secretions need to be further verified.
Total organic acids were secreted the most at 1 and 5 mg/L, and decreased
significantly at 20 mg/L phosphorus concentration, indicating that the
phosphorus concentration most suitable for the growth of H. verticillata
may be in the 1–5 mg/L range. Phosphorus concentrations above or below that
range may have been far less or more than the amount needed by the H.
verticillate, respectively.
High phosphorus concentrations can destroy plant root tissue and lead to a
decline in plant root exudation. Phthalic acid is the main organic acid
secreted by the root system of H. verticillata at all culture levels,
which is different from the previous studies on plants such as Brassica
napus and soybean in response to phosphorus control stress, wherein the
main acid secreted is malic acid (Duan 2003; Zhang et al. 2011), while Clove secretes citric acid, malic acid,
and succinic acid (Montague 1984). Broussonetia papyrifera and Orychophragmus
violaceus primarily secrete oxalic acid, citric acid, and malic acid (Zhao
and Wu 2014).
Plant diversity might explain the reason underlying the secretion of
various acids. H. verticillata is a typical wetland submerged plant,
different from the terrestrial plant growth environment. The concentrations of
phosphorus stress were phosphorus deficiency, low concentration and high
concentration of phosphorus, and the relative content of phthalic acid was the
highest in the organic acids secreted under different culture time. This
indicates that the high secretion of phthalic acid is the adaptation mechanism
of diatoms in wetland submerged plants under the stress of external nutrient elements.
Conclusion
Under phosphorus stress for 7 days,
the least amount of carbonic acid, phthalic acid, phenyldicarboxylic acid,
benzoic acid, and total organic acid was observed. With the increase in
phosphorus concentration, the secretion of benzoic acid and phthalic acid
initially decreased followed by an increase. With the increase in phosphorus
concentration, the secretion of benzoic acid and phthalic acid increased first
and then decreased. Oxalic acid secretion gradually decreased with the increase
in phosphorus concentration, and the amount of carbonate, phthalic acid,
benzene dicarbonic acid, and benzoic acid gradually increased with the increase
in phosphorus stress concentration. On days 14, 21 and 28, the least secretion
of phthalic acid, benzene dicarbonic acid, and total organic acid was observed
in the condition of no phosphorus stress, whereas sulfurous acid, carboxylic
acid, sulfonic acid, and benzene dicarbonic acid secretion gradually increased
with the increase in phosphorus stress concentration. The results indicate that
the secretion of organic acids in the roots of H. verticillata is
closely related to the concentration of phosphorus stress and the duration of
stress period. Phosphorus stress causes the roots to adjust the secretion of
organic acids actively. Phthalic acid is the main organic acid secreted by H.
verticillata roots at all culture levels, indicating that the regulation of
phthalic acid secretion by the root system under phosphorus stress is an
important mechanism of active adaptation to environmental changes.
Acknowledgements
National Science Foundation of
China (No. 31760149;31860235), Fund to Key Research and Development Program of
Yunnan Provincial (No.2018BB018), Fund Project to The forestry science and
technology innovation platform project of The State Forestry Administration
(2019132161; 2019-YN-13).
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