Non-cropping period accounting for over a half of annual nitric oxide releases from cultivated calcareous-soil alpine ecosystems with marginally low emission factors

ABSTRACT Nitric oxide (NO) emissions from alpine ecosystems conventionally being long-term cultivated with feed crops are not well quantified. The authors attempted to address this knowledge gap by performing a year-round experimental campaign in the northeastern Tibetan Plateau. Fertilized (F) and unfertilized (UF) treatments were established within a flat calcareous-soil site for the long-term cultivation of feed oats. NO fluxes and five soil variables were simultaneously measured. A single plow tillage accounted for approximately 54%–73% of the NO releases during the cropping period (CP); and the non-cropping period (NCP) contributed to 51%–58% of the annual emissions. The direct NO emissions factor (EFd) was 0.021% ± 0.021%. Significantly lower Q10 values (p < 0.01) occurred in the F treatment during the CP (approximately 3.6) compared to those during the other period or in the other treatment (approximately 4.9−5.1), indicating a fertilizer-induced reduction in the temperature sensitivity. The selected soil variables jointly accounted for up to 72% (p < 0.01) of the variance for all the fluxes across both treatments. This finding suggests that temporally and/or spatially distributed fluxes from alpine calcareous-soil ecosystems for feed crop production may be easily predicted if data on these soil variables are available. Further studies are needed to test the hypothesis that the EFd is larger in alpine feed-oat fields than those in this study if the soil moisture content is higher during the period following the basal application of ammonium- or urea-based fertilizer. Graphical Abstract

amended with nitrogen only in form of organic nitrogen in the oat seeds (8 kg N ha −1 ).
Four days before the campaign period, four replicated field plots (each with a size of 55 m 2 for the F and 1010 m 2 for the UF) for each treatment were randomly established within the select land. Each plot was situated with at least a 2-m distance between the closest boundaries of a plot and the select land and at least a 5-m distance between the closest boundaries of any two plots of the two treatments. Such layouts of the field plots were chosen to minimize the effects of plot/land boundaries and spatial variabilities in soil properties, vegetation and anthropogenic activities, thus facilitating statistical comparisons of the experimental results between the two treatments. The exact location and area of each field plot were fixed during the entire campaign period. As required for field experiments to determine a direct nitrous oxide or nitric oxide (NO) emission factor (IPCC 2006), the UF plots had previously received the same fertilization practices as the F areas but were free from amendment of the synthetic fertilizers only in the year-round experimental period.
Four days before the first observation of NO fluxes, one chamber base frame was installed in the center of each replicated field plot. The base frame remained permanently in each field plot, except for temporary removal and reinstall to allow for soil plowing and follow-up mechanical operations. Each frame was made of stainless steel (each 50 cm long, 50 cm wide and 15 cm high; each wall was 3.0 mm thick) and was inserted fully into the soil; only the upper-edge collar extended out of the soil surface. A rubber band (6 mm thick) was applied to the upper-edge collar of each base frame for gas-tight sealing of the joint with the chamber. Each chamber was made of stainless steel (50 cm long, 50 cm wide and 40 cm high; each wall was 1.0 mm thick; no bottom). The walls were coated with polystyrene foam boards that were covered with tinfoil to minimize the temperature change within the headspace enclosure during gas sampling. When the vegetation was taller than 40 cm, an alternative chamber with an 80 cm height was adopted to avoid physical damage to the plants within each base frame.
There was a tube (7.4 mm inner diameter and 12 cm length) on the top wall of each chamber to allow for an air connection between the headspace and the atmosphere to minimize the pressure difference during sampling. To measure the flux from each plot, a chamber was temporarily mounted onto one of the two base frames to establish a 10-min headspace enclosure for gas sampling.
For the convenience of arranging temporarily intensified observations, a freeze-thaw period (FTP) was defined as a period of at least 5 d during which the daily mean air temperature consecutively fell within the range from −10 to 0 °C. Accordingly, the spring FTP occurred during the period from 21 February to 21 April, 2014 (Fig. S1).
The period from the sowing date to the harvest date, the total remaindering time of the full campaign duration, and the full year-round campaign period are referred to as the cropping period, the non-cropping period, and the annual period, respectively.

B. Measurement of nitric oxide fluxes
Nitric oxide fluxes from each field plot were measured during the entire campaign using a technique of combining the chemiluminescence analysis of NO concentrations with gas sampling by opaque, static chambers (Mei et al. 2009;Liu et al. 2009Liu et al. , 2015Zhang et al. 2018). The flux measurements for all field plots were manually performed daily or every other day during the spring FTP and the periods of 1014 d following plowing and fertilizing; otherwise, the measurements were performed once every 34 d.
The methods of gas sampling, instrument calibration, and analyses of both NO and nitrogen dioxide (NO 2 ) concentrations in the gas samples as well as the flux calculations were in accordance with those detailed by Zhang et al. (2018). The sample NO that was collected into a gas bag until analysis could be converted partly into NO 2 in the presence of ozone. In this regard, the sum of the simultaneously measured NO and NO 2 fluxes was regarded as the measured NO flux. A single NO flux measured by gas sampling during the local time 08:0010:00 a.m. was used to represent the daily average value (e.g., Liu et al. 2010). According to the instrument precision for NO or NO 2 analysis (0.3 nmol mol 1 for both gases) and the enclosure time (10 min), the detection limits of NO fluxes for the adopted chamber heights of 40 and 80 cm were 0.4 and 0.8 μg N m −2 h −1 , respectively.
It should be noted that the NO fluxes measured in this study represented only conservative magnitudes for the investigated ecosystems. The reason is the applied method with a linear-change assumption regarding the gas concentrations in individual static, opaque chamber enclosures might significantly underestimate NO fluxes, e.g., by approximately 31% (ranging 3%59% at the 95% confidence interval), in comparison with a nonlinear approach (Mei et al. 2009;Yao et al. 2015). The underestimations due to a linear-change assumption indeed do not allow accurate quantification of area-scaled or yield-scaled NO emissions. Nevertheless, the high sensitivity of the applied method to measure NO fluxes allowed investigations of the differences between field treatments and thus determination of direct NO emission factors of applied fertilizer nitrogen (Yao et al. 2015). The high sensitivity also allowed investigation of the regulatory effects of soil variables and other factors on NO fluxes (e.g., Zhang et al. 2018).

C. Auxiliary measurements
The air pressure, air temperature and precipitation were observed and provided by the HAMERS. When the NO fluxes were measured, air temperature within chamber headspace enclosure, topsoil (5 cm depth) temperature (T s ) and surface (06 cm depth) soil moisture in water-filled pore space (WFPS) were simultaneously measured. The air pressure and headspace air temperature were observed since both variables are involved in the flux calculation to correct the NO gas density. The concentrations of soil (010 cm depth) ammonium (NH 4  ), nitrate (NO 3  ) and water-extractable organic carbon (WEOC) were observed weekly on one of the days when the NO fluxes were measured. Select soil properties of the (020 cm depth), including clay (< 0.002 mm), silt (0.002-0.05 mm), and sand (0.05-2 mm) fractions, soil organic carbon (SOC) and total nitrogen (TN) contents, and soil pH, were measured once in mid-autumn. The mean values were presented for the surface soil (06 cm depth) bulk density (BD) that was seasonally measured. The aboveground biomass, which was regarded to approximate the aboveground net primary productivity, and its nitrogen content were also measured at harvest.
To measure the aboveground biomass and its nitrogen content, the harvested plant materials were oven-dried for 30 min at 105 °C and then for 48 h at 60 °C to obtain the dry matter weight. The nitrogen contents in the dried plant samples were analyzed using the Kjeldahl method (Bao 2000).
The SOC and TN concentrations were analyzed using the potassium dichromate oxidation and the Kjeldahl methods, respectively (Bao 2000). A water-to-soil ratio of 2.5 was used to determine the pH values. The soil particle fractions of different size ranges were measured using Malvern laser particle analysis (Yang et al. 2009).
The headspace air temperature and T s were manually measured during gas sampling using digital thermal couples.
The topsoil (06 cm) volumetric water content ( v , cm 3 cm 3 ) was manually measured during the unfrozen periods using a portable frequency-domain reflector moisture meter. During the frozen periods or FTPs, the gravimetric water contents ( w , g g 1 ) in both ice and liquid phases were measured by oven-drying the soil sample and then the values were converted to  v units by multiplying with BD (g cm 3 ). Finally, each soil moisture content in WFPS was calculated as WFPS = 100 v /(1 BD/2.65).
At each time, four samples were collected (each was a mixture of soil samples from five random points within the corresponding field plot), well mixed, sieved with a 2-mm mesh, and ultimately subsampled via three replicates. On the same day, the subsamples were extracted for analysis of NH 4  , NO 3  (1 M potassium chloride, solution-to-soil ratio = 5; shaking for 1 h, and filtering by filter paper) and WEOC (distilled water, water-to-soil ratio = 5; shaking for 1 h, centrifuging for 10 min at 6000 rpm, and filtering by polyethersulfone membrane with  0.45μm pores) concentrations. Each extract was saved in a 50-mL polyethylene-terephthalate bottle at approximately 18 °C for later assay. The concentrations of NH 4  , NO 3  and WEOC in the extracts were analyzed shortly after thawing for 24 h at 4 °C using a continuous flow analyzer (San  , Skalar Analytical B.V., The Netherlands).

D. Data analysis and statistics
The bivariate correlation analysis method was adopted to test the correlations of the The standard errors of means for three to five spatial replicates were given to report the results if not otherwise specified.

Table S1
Select soil properties and other natural or management information of the experimental site and the fertilized (F) and unfertilized (UF) field treatments.  (1) 36 (1)  49(1) Clay (< 0.002 mm) fraction (%) a,b,c 17 (1) 15 (1) Organic carbon content ( replicates. c and d , for 020 and 06 cm soil depths, respectively. For the full treatment names, refer to Table S1. FPT and CP were from 21 February to 21 April, and 2 June to 26 September, 2014, respectively.
The given data are means of 3-5 spatial replicates, with standard errors showing within the parentheses. The different superscript capital letters indicate the significant differences between CP and NCP within a treatment at p  0.05, respectively.   Table S2. r 2 , n, p and Q 10 denote the coefficient of determination, sample size, significance level, and temperature sensitivity coefficient, respectively. A given Q 10 value represents the fold of the changes in NO fluxes due to a 10-degree change in soil temperature.