2.4 | Sample collection and determination
At the end of the culture experiments, the roots were washed with
deionized water and blotted dry with absorbent paper, and the weights of
the plants’ above- and below-ground parts were measured separately. Two
boxes of culture substrates in each group were randomly combined into
one sample, mixed and sampled by quartering. Pass through a 40-mesh
sieve after grinding to determine soil enzymes and polysaccharides.
Enzymatic activities of S-UE, S-NiR, S-NR, and S-CAT were evaluated
using cominbio kits (Suzhou Keming Biotechnology Co., Ltd.) following
the manufacturer’s guidelines, while mlbio kits (Shanghai Enzyme
Biotechnology Co., Ltd.) were employed to measure the activity of S-PT
enzymes. The determination of pentoses adopts the lichenol-hydrochloric
acid method (Haoli et al., 2014), and the determination of hexoses
adopts the anthrone-sulfuric acid method (wei-jie, 1999), and the sum of
the two is calculated as the total polysaccharide content.
S-NiR activity was assessed using the cominbio kit’s method. The amount
of reducing 1 μmol NO2ˉ per g soil sample per day was
regarded as an enzyme activity unit. In a 2 mL centrifuge tube, 0.02 g
of air-dried soil sample was mixed with 40 µL each of sodium nitrite and
glucose solutions. The mixture was thoroughly combined and reacted for
one hour at 25℃. Subsequently, 40 µL of aluminum potassium alum solution
was added, and the content was fully agitated for thirty seconds before
centrifugation was facilitated at 10,000 rpm, at 4℃, for ten minutes. At
this stage, 70µL of the supernatant was extracted, along with 1:1
p-amino benzene sulfonic acid-phosphoric acid solution and 140 µL of
N-1-naphthalene ethylenediamine hydrochloride solution. The components
were mixed well and evaluated for absorbance at 540 nm. Control tubes
were supplied with distilled water instead of sodium nitrite solution,
while blank tubes had no samples. S-NR activity was assessed using the
cominbio kit’s method, and the amount of 1 μmol NO2ˉ
produced per gram of soil sample per day was regarded as one S-NR
activity unit. Air-dried soil samples weighing 0.06 g were included in a
2 mL centrifuge tube containing 225 µL of KNO3 solution
and 75 µL of Reduced Coenzyme I (NADH) solution. Samples were then
subjected to a 24-hour water bath at 37℃ before centrifugation at 8000g
and 25℃ for 10 min. At this stage, 130 µL of the supernatant was
collected and added to 85 µL each of p-amino benzene sulfonic acid
solution and α-naphthylamine solution. The resulting mixture was
agitated and left for color development at 25℃ for 20 min before
centrifugation at 4000g for 10 min at 25℃. 200 µL of the supernatant was
transferred to a 96-well plate and monitored for absorbance at 540 nm.
Distilled water replaced the KNO3 solution for the
control tube, while the blank tubes omitted soil samples. The standard
tube replaced the soil sample with 0.1 μmol/mL NaNO2solution. S-UE activity was assessed using the cominbio kit’s method,
and 1 μg of NH3-N produced per gram of soil sample per
day was defined as an enzyme activity unit. Air-dried soil samples
weighing 0.06 g were placed in 2 mL centrifuge tubes after adding 20 µL
of toluene and shaking, then placed at room temperature for 15 min.
Afterward, 90 µL of urea and 190 µL of citric acid-potassium hydroxide
solution were blended before a 24 h water bath at 37℃. Following this,
centrifugation was carried out at 10,000g and 25℃ for 10 min. The
supernatant was then diluted ten-fold, with 80 µL taken, and the mixture
was coupled with 15 µL of phenol, methanol, acetone, absolute ethanol,
and NaOH solutions. Next, 15 µL of sodium hypochlorite solution was
added and mixed well before being placed at room temperature for 20 min.
The absorbance of the resultant mixture was monitored at 578 nm after
the inclusion of 90 µL of distilled water. Distilled water substituted
urea in the control tube. S-CAT activity was assessed using the cominbio
kit’s method, and the degradation of 1 μmol
H2O2 catalyzed per g of air-dried soil
sample per day was defined as an enzyme activity unit. Air-dried soil
samples weighing 0.03 g were included in a 2 mL centrifuge tube and
supplemented with 260 µL of hydrogen peroxide. Samples were subjected to
a 20 min incubation at 25℃ while agitated at 500r/min. Next, 10 µL of
aluminum potassium alum was added, followed by centrifugation at 8000g
and 25℃ for 5 min. 180 µL of the supernatant was collected and mixed
with 20 µL of sulfuric acid solution before monitoring absorptivity at
240 nm. The control tube contained distilled water instead of hydrogen
peroxide, while the blank tubes contained no soil samples. S-PT activity
was assessed using the mlbio kit’s method. To prepare the soil samples
for testing, 0.1 g of soil was mixed with 0.9 mL of Phosphate buffer
saline (PBS) before centrifugation at 4000r for 15 min. The resultant
supernatant became the sample for examination. Next, 50 µL of the
diluted standard and 40 µL of the sample diluent were added to the
sample, which was then sealed with a sealing film and incubated at 37℃
for 30 min. Afterward, the liquid was discarded, followed by the
introduction of wash solution, left for 30 s, and discarded five times.
Subsequently, 50 µL of HRP enzyme-labeled reagent was included, and
incubation and washing processes resumed before adding 50 µL of
carbamide peroxide color reagent and 50 µL of color reagent. At this
stage, color development was initiated, with 10 min allotted for
completion in the dark at 37℃. Additionally, 50 µL of termination
solution was infused before absorbance levels were measured at 450 nm.
Blank wells that omitted samples and enzyme labeling reagents were also
featured. Phosphotransferase markers (Figure. S1). The content
determination method of the total polysaccharide in the soil adopts the
method of pansu et al. (Pansu and Gautheyrou, 2006). Preparation of the
test solution: weigh 2.5g of air-dried soil, put it in a 50mL Conical
flask, then add 20 mL of 2.5M H2SO4,
heat and reflux in a water bath at 100℃ for 20 min, let cool, filter,
wash, and collect the filtrate. Put the residue in a 50mL Conical flask,
add 1mL 13M H2SO4, and place it at room
temperature for 16 h. Slowly add 25 mL of distilled water, let cool,
heat at 100℃ for 5 h after airtight, let cool, filter, combine the
filtrate with the above filtrate and dilute to 50mL to obtain the test
solution. The total polysaccharide content was determined using the
lichenol-hydrochloric acid method for pentose, while hexose was assessed
using the anthrone-sulfuric acid method. Pentose and hexose markers,
respectively (figure s3, figure s2). Finally, the sum of the two was
calculated as the polysaccharide content.