DOI: 10.5061/dryad.63xsj3v0h
title: Data from: Differential impacts of nitrogen addition on rhizosphere and bulk-soil carbon sequestration in an alpine shrubland

description: 1. Due to complex root-soil interactions, the responses of carbon (C)
 dynamics in the rhizosphere to elevated nitrogen (N) deposition may be
 different from those in bulk soil. However, the potentially different
 response of C dynamics in the rhizosphere and bulk soils and their
 contributions to soil C sequestration under N deposition is still not
 elucidated. 2. We conducted an N addition experiment in an alpine
 shrubland dominated by Sibiraea angustata located on the eastern
 Qinghai-Tibet Plateau (QTP). We measured the soil organic C (SOC) contents
 and density fractions in the rhizosphere and bulk soils in the top 15 cm
 of mineral soil and then employed a numerical model based on the
 rhizosphere extent to evaluate how the rhizosphere modulates soil C
 sequestration under N addition. We also measured the microbial gene
 abundance and C-acquisition enzyme activities to assess microbial
 community responses to N addition. 3. The results showed that nitrogen
 addition had opposite effects on the rhizosphere and bulk-soil C stocks.
 Specifically, N addition decreased the rhizosphere SOC content through
 increasing bacterial abundance, β-glucosidase activity, and thus
 accelerating the loss of free light fraction C (FLF-C). However, N
 addition increased the bulk-soil C content, which was corresponding with
 the reduced oxidase activities and the accelerated accumulation of heavy
 fraction C (HF-C) under N addition. Numerical model analysis showed that
 the decrease induced by N addition in rhizosphere SOC stock ranged from
 0.11 to 3.01 kg C m-2 as root exudation diffusion distance extended from
 0.5 mm to 2 mm, while the corresponding increase in the bulk-soil C stock
 ranged from 1.91 to 4.08 kg C m-2. By synthesizing the dynamics of the SOC
 stocks in these two soil compartments under N addition, the SOC stock at
 the ecosystem level exhibited an increase in range of 0.73-2.44 kg C m-2.
 4. Synthesis Our results suggest that alpine shrublands on the
 eastern QTP have great potential for soil C sequestration under N
 deposition, and the magnitude of the sequestration would depend closely on
 the responses of rhizosphere microbial C processes and the rhizosphere
 extent. Our results highlight the importance of integrating rhizosphere
 processes into land surface models to accurately predict ecosystem
 functions in the background of elevated N deposition.
description: 1. Study site: The Field Research Station of Alpine Shrubland
 Ecosystem is located in Songpan County, Sichuan Province, China. This
 region belongs to a typical alpine climate. The community composition of
 plants was dominated by temperate and cold-temperate vegetation at our
 study region. The dominant shrub species was Sibiraea angustata, followed
 by Salix oritrepha, Spiraea alpine, and Potentilla fruticosa (Wang et al.,
 2017). The average coverage, basal diameter and height of Sibiraea
 angustata are 53%, 0.62 cm and 1.03 m, respectively. Our previous study
 showed that the aboveground and belowground biomasses of Sibiraea
 angustata were 3.71 kg m<sup>-2</sup> and 4.39 kg
 m<sup>-2</sup>, respectively, accounting for 96% and 97% of
 the aboveground and belowground biomass (Wang et al., 2017). These results
 suggest that both the aboveground- and belowground-biomass of Sibiraea
 angustata have an overwhelming superiority within this shrub community.   
                                                                          
                                 2. The manipulated
 experiment of nitrogen addition: An experiment of nitrogen addition was
 initiated within this shrubland ecosystem in May 2012. A randomized block
 design with three replicated blocks of three treatments was established
 before the experiment; these treatments included the control (no N
 fertilizer), low-N addition (50 kg N ha<sup>-1</sup>
 year<sup>-1</sup>), and HN (100 kg N
 ha<sup>-1</sup> year<sup>-1</sup>). Each block
 included three plots in 5 m × 5 m, and each plot was surrounded by
 10-m-wide buffer strips. According to the average increase of bulk N
 deposition across China (approximately 8 kg N ha<sup>-1</sup>
 yr<sup>-1</sup>; Liu et al., 2013) and the level of N
 fertilizer experiments performed in neighbor sites on the QTP (10 and 350
 kg N ha<sup>-1</sup> year<sup>-1</sup>; Fu et al.,
 2017), we chose the doses of the N additions in our study equal to be
 nearly 3- and 5-times the value of the background N deposition. An
 ammonium nitrate (NH<sub>4</sub>NO<sub>3</sub>)
 solution has been sprayed onto the forest floor in 6 equal doses in the
 first week of every month from May to October (i.e., the growing season)
 since May 2012. In each N-addition event, the fertilizer was weighed,
 dissolved in 20 L of water, and sprayed evenly using backpack sprayers.
 The control plots received an equal volume of water. 3.
 Soil sampling protocol: We sampled the upper 15 cm of mineral soil in July
 2017. After organic horizon removal, five replicate cores from each plot
 using a 6-cm diameter soil corer to ensure that fine roots would have a
 sufficient mass of adhering rhizosphere soil. The soil cores were stored
 on ice transported to the laboratory. The living roots of Sibiraea
 angustata in each core were empirically identified by features such as
 shape, color, and elasticity. The soil adhering to the roots was carefully
 separated from the roots using fine forceps; this fraction was
 operationally defined as rhizosphere soil, and non-adhering soil was
 considered to be bulk soil (Phillips et al., 2011). All soil samples were
 passed through a 2-mm mesh sieve and were divided into two parts: one part
 was used to determine the soil physicochemical properties and the soil
 carbon density fractionation; another part was stored at -20 °C for later
 analyses of microbial biomass carbon and enzymatic activity.
 4. Basic physicochemical properties: For soil bulk density (SBD)
 determination, a bulk-density corer with 5-cm diameter stainless steel
 rings as an inner sleeve was manually inserted into the upper 15-cm depth
 of the mineral soil (three corers for each plot) (Davidson et al., 2004).
 The SOC was determined by dichromate oxidation and titration with ferrous
 ammonium sulfate (Walkley and Black, 1934). The DOC was extracted with 0.5
 M K2SO4 and determined using a TOC analyzer (Vario TOC, Elementar Corp.,
 UK). The total N concentration (SON) in soil was determined on a TOC
 analyzer (Vario TOC, Elementar Corp., UK). Soil
 NH<sub>4</sub><sup>+</sup> and
 NO<sub>3</sub><sup>-</sup> were extracted by 2 M
 KCl (soil:solution = 1:5) and then determined on a continuous flow
 injection analyzer (SEAL Analytical, Germany). The dissolved inorganic N
 (DIN) was the sum of NH<sub>4</sub><sup>+</sup>
 and NO<sub>3</sub><sup>-</sup>. The soil organic N
 concentration (SON) in soil was the difference between TSN and DIN.
 Available phosphorus (Av.P) was extracted with Bray-I solution (0.03 M
 NH4F - 0.025 M HCl) (Bray and Kurtz, 1945) and was determined by
 molybdenum antimony colorimetry. Soil pH was measured in slurry with a
 soil-to-water ratio of 1:2.5 using a pH meter (Mettler-Toledo Instruments
 Co., Ltd., Shanghai, China). 5. Soil carbon
 fractionations: The SOC fractions were separated using a density fraction
 method (McLauchlan and Hobbie, 2004). Briefly, a 15 g of air-dried
 rhizosphere or bulk soil (passed through 2mm-mesh sieve) was weighed and
 placed in a 100 mL centrifuge tube and dispersed in 50 mL of NaI (with a
 density of 1.8 g cm<sup>−3</sup>). The tubes were centrifuged
 at 3000 rpm for 20 min. The suspended materials (FLF) were decanted into a
 vacuum filter unit with 0.40 µm polycarbonate filter. This process was
 repeated 2-3 times until no floating material remained. The materials
 remaining at the bottom (HF) of the centrifuge tube were then rinsed into
 the vacuum filter unit. All samples on the filter paper were washed with
 75 mL 0.01 mol L<sup>−1</sup> CaCl<sub>2</sub>,
 followed by at least 75 mL of distilled water. The light and heavy
 materials were dried at 60°C for 48 h and weighed. All samples were passed
 through a 0.25 mm mesh sieve and analyzed for SOC as previously
 described. 6. Microbial gene abundance: For microbial
 gene abundance, DNA was extracted from 0.25 g of soil using the MoBio
 Power Soil DNA isolation kit (Mobio Laboratories, CA, USA). DNA quality
 and concentration were measured using a nanodrop spectrophotometer
 (NanoDrop, DE, USA) and electrophoresis in agarose gels (1% w/v in TAE),
 then stored at -20 °C prior to amplification. Quantitative PCR (qPCR) was
 used to quantify the gene copy numbers of bacterial 16S rRNA and fungal
 ITS using the primer pairs 515F/909R and ITS7F/ITS4R, respectively (Li et
 al., 2014; Schulz et al., 2018). Each 10-µL reaction contained 5 µL of
 SybrGreen (2×) PCR Master Mix (Bio-Rad, USA), 0.5 µL of each primer (10
 pM), 2 µL of DNA templates and 2.5 µL of sterilized water. Bacterial 16S
 rRNA and fungal ITS conditions were 5 min at 95 °C, followed by 40 cycles
 of 95 °C for 30 s, 30 s at 55 °C, and 30 s at 72°C, with a final extension
 cycle of 8 min at 72°C. The qPCR standards for quantification were
 prepared from PCR products of target genes from environmental DNA with
 each primer set using the method described by Kou et al. (2017). Four
 replicates were performed for each sample. The amplification efficiencies
 of the 16S rDNA gene and ITS gene were 90% and 92%, respectively, with
 R<sup>2</sup><sub> </sub>values higher than 0.99,
 and no signals were observed in the negative controls.
 7. Carbon-acquisition enzyme activities: Two grams of sieved soil
 was suspended in 125 mL of 50 mM sodium acetate buffer (pH = 5.0) in a
 homogenizer for 1 min to form slurry. Black 96-well microplates were used
 for fluorometric analysis. The microplates were assigned to six parts,
 including the sample assay, sample control, quench control, reference
 standard, negative control, and blank wells. First, 200 μL of buffer was
 pipetted into the blank, reference standard and negative control wells.
 Second, 50 μL of buffer was pipetted into the blank and sample control
 wells. Third, 200 μL of the soil slurry was pipetted into the sample
 assay, sample control, quench control wells, and then 50 μL of 10 μM
 4-methylumbelliferyl (MUB) was pipetted into the reference standard and
 quench standard wells. Finally, 50 μL of 200 μM fluorogenic substrate
 (4-methyl-umbelliferyl β-D-glucopyranoside) were pipetted into into the
 negative control and sample assay wells. Plates were incubated for 4 h in
 the dark (25°C) and then scanned on a Varioskan Flash multiplate reader
 (Thermo Scientific, USA) at 365 nm excitation and 450 nm emission
 wavelengths. The unit for BG activity was expressed as μmol MU
 g<sup>-1</sup>dry soil (dry weight)
 h<sup>-1</sup>. Phenol oxidase and
 peroxidase activities were measured spectrophotometrically
 using <sub>L</sub>-3, 4-dihydroxyphenylalanine
 (<sub>L</sub>-DOPA, Sigma, St. Louis, USA) as the substrate. A
 total of 200 μL soil suspension (see above) and 50 μL of 25 mM DOPA were
 added to each sample well. The wells of peroxidase assays additionally
 received 10 μL of a 0.3%
 H<sub>2</sub>O<sub>2</sub> solution. The
 microplates were incubated in the dark at 20°C for up to 8 h. Absorption
 was measured at 450 nm and expressed in units of μmol DOPA
 g<sup>−1</sup> h<sup>−1</sup>. The background
 absorbance of DOPA was measured, and an extinction coefficient was
 calculated using a standard curve of DOPA degraded with mushroom
 tyrosinase.