N2O flux measurements
In 2013, a 40 cm ×40
cm
square stainless steel collar was permanently inserted into the topsoil
(~ 10 cm), which located in the dynamic monitoring area
of each plot. The in-situ N2O flux was measured using
static chamber with insulation materials and gas chromatography
techniques. During gas collection (between 8 am and 12 noon),
a
chamber (30 cm tall) with an electric fan (to mix the air) was placed on
the collar. Gas samples (100 mL)
were collected by medical syringes at intervals of 0, 10, 20 and 30 min
and then promptly injected into multi-layer foil sampling bags (Delin
Inc., Dalian, China). In 2020, we
collected gas samples three times per
month (May to October). Furthermore, we conducted that gas samples
collection during six consecutive days in mid-August
(plant growth peak). The collected
gas samples were immediately transferred to the laboratory and then
determined for N2O concentration using a GC-7890B gas
chromatograph (Agilent Technologies Limited Co., Chengdu, China). While
collecting gas (plant growth peak), the soil volumetric water content
(VWC) and temperature in the top 10 cm were measured in each plot
adjacent to the collar using a hand-held moisture probe and a digital
thermometer, respectively. The N2O flux was calculated
as follows:
F= \(\ \rho\times\frac{V}{A}\)×\(\frac{T0}{T}\)×\(\frac{P}{P0}\)×\(\frac{\text{dc}}{\text{dt}}\)
where F is the N2O flux (µg N2O
m−2 h−1); ρ is the standard
status N2O density; V is the volume of the static
chamber (m3); and A is the base area of the
static chamber (m2). T 0 and T are the
standard temperature (273 K) and the static chamber temperature (K),
respectively. P 0 and Pare the standard pressure (1,013 hPa) and the air pressure (hPa),
respectively. The rate of increase in the N2O
concentration in the static chamber (10−6h−1) is dc/dt .
Soil and plant sampling and chemicalanalyses
To identify the mechanisms regulating N2O flux responses
to N input and altered precipitation,
plant and soil samples were collected at the peak of plant growth.
First, three 25 cm × 25 cm quadrants were randomly placed in each plot,
and then all living plants were clipped as aboveground biomass. After
removal of the aboveground plants, three root cores
(internal
diameter 8 cm and depth 10 cm) were collected and then mixed. The mixed
root cores were washed with water in a 0.4 mm sieve.
The
live roots were selected by their color and were used as
belowground
biomass. The
collected
aboveground and belowground biomasses were oven-dried at 60°C to a
constant mass and were then weighed.
Three more soil cores (internal
diameter 3 cm and depth 10 cm) were collected near each collar (for a
total of 90 soil cores) and were then homogenized to acquire one
compound sample (for a total of 30 soil samples). The collected soil
samples were separated into three subsamples by a sieve (2 mm). The
first subsample was immediately preserved at −80°C for DNA extraction
and also analysis of the abundances of key microbial functional genes.
The second subsample was stored at 4°C to determine the soil ammonium
(NH4+-N) and nitrate
(NO3−-N)
concentrations. The third subsample
was air-dried to determine the soil pH. The available N
(NH4+-N and
NO3−-N) concentrations in soil were
determined using a flow injection analyzer (Autoanalyzer 3 SEAL, Bran
and Luebbe, Norderstedt, Germany) after extracting fresh soil with 1 M
KCl solution. The pH of the air-dried soil was measured using a pH
electrode (soil-to-deionized water ratio of 1:2.5).
Soil
DNA extraction and real-time quantitative PCR (qPCR)
Soil DNA was extracted from 0.5 g frozen soil using a kit (E.Z.N.A.® DNA
Kit, Omega Bio-tek, Norcross, GA, U.S.A.) based on the manufacturer’s
instructions. The DNA extract was
checked on 1%
agarose
gel.
The quality of the DNA was evaluated with a NanoDrop 2000 UV-vis
spectrophotometer (Thermo Scientific, Wilmington, DE, U.S.A.).
The
nitrification-related amoA gene in ammonia-oxidizing bacteria
(AOB) and archaea (AOA) was determined. The nirS , nirK ,
and nos Z genes, which are associated with denitrification, were
also determined in denitrifying microorganisms. The functional gene copy
numbers were amplified using an ABI 7300 Real-Time PCR System (ABI, CA,
U.S.A.). PCR reactions were
performed in triplicate. The PCR mixtures contained 10 μL 2X ChamQ SYBR
Color qPCR Master Mix, 0.8 μL forward primer 5 (μM), 0.8 μL reverse
primer (5 μM), 2 μL template DNA, 0.4 μL 50 X ROX Reference Dye 1, and 6
μL ddH2O. The functional genes, primers, and sequences
used for PCR reactions are summarized in Table 1. More detailed PCR
thermal cycling conditions are listed in Table S1. The standard curve of
each amplified gene was constructed using a 10-fold dilution of plasmid
DNA (containing the target gene). The PCR efficiency was between 89%
and 101%; the R2 ranged from 0.98 to 0.99.