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.