LIN Fei,LIU Chun-Yn,HU Xio-Xi,FU Yong-Feng,ZHENG Xunhu,ZHANG Wei,WANG Rui nd CAO Gung-MinStte Key Lortory of Atmospheri Boundry Lyer Physis nd Atmospheri Chemistry,Institute of Atmospheri Physis,Chinese Ademy of Sienes,Beijing,Chin;College of Erth nd Plnetry Sienes,University of Chinese Ademy of Sienes,Beijing,Chin;Reserh Center for Plteu Eology,Northwest Institute of Plteu Biology,Xining,Chin

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 Q10values(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 EFdis 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 ammoniumor urea-based fertilizer.

KEYWORDS Nitric oxide(NO)emissions;direct NO emissions factor;alpine meadow cultivation;non-growing season;freeze–thaw period;plow tillage effect;temperature sensitivity(Q10)

1.Introduction

Nitric oxide(NO)is a precursor for generating very important air pollutants,such as secondary aerosol particles to form haze,nitric acid to form acid rain,and tropospheric ozone(Atkinson et al.2004).As such,NO clearly contributes to both air pollution and climate change.Terrestrial ecosystems,after the combustion of both fossil fuels and biomass,play an important role in atmospheric NO sources.In particular,soils are usually the predominant atmospheric NO source in remote regions(e.g.,Bouwman,Boumans,and Batjes 2002).In this regard,NO emissions from fertilized ecosystems in alpine regions,e.g.,the Tibetan Plateau,are important to understand and quantify.

Pasture dominates the grazing-based agriculture in temperate alpine regions.Nevertheless,long-term cultivation of some of the grasslands to grow feed crops has conventionally occurred widely on the Tibetan Plateau and elsewhere.This has long been a practical solution of the feed shortage problem for grazing livestock during the long and cold winters(e.g.,Zhao 2004).For this reason,the grassland landscapes on the Tibetan Plateau have been modified greatly since the 1960s,mainly due to intensified heavy grazing as well as cultivation of alpine crops(Chen et al.2013).Oat(Avena sativa)has long been the most important crop of the Tibetan Plateau and accounts for up to 70%of the total feed crop area of the region(Zhao et al.2004).However,the characteristics of NO emissions during the widely adopted practice of feed oat cultivation remain unknown.In particular,quantification of the direct NO emissions factor(EFd)of the nitrogen fertilizers consumed during feed crop cultivation in alpine regions is still lacking.This situation may greatly bias the NO emissions inventories of alpine regions,because the EFdvalues observed in non-alpine regions,which most likely do not apply in alpine regions,have to be used instead.The present reported study was an attempt to narrow this knowledge gap.

In this study,we launched a year-round field experimental campaign in a typical feed-oat cultivation site located in the northeastern Tibetan Plateau.The experimental campaign aimed to(i)establish a field experiment for the purpose of determining the EFdfor the typical fertilization practices of feed-oat cultivation in the Tibetan Plateau region;(ii)simultaneously observe the NO fluxes and other related variables of the established field treatments;(iii)identify the differences in the NO emissions between the field treatments and understand their regulatory mechanisms;and(iv)provide EFdmeasurements.These efforts were conducted to test the hypothesis that the EFdvalues obtained in non-alpine regions are too high,and thus not applicable in estimating the inventories of feed-crop cultivation in alpine regions.

2.Materials and methods

The selected field site(37°36′45″N,101°18′48″E;3203 m above sea level)was located aside the Haibei Alpine Meadow Ecosystem Research Station(HAMERS),which is situated in Qinghai Province,China.The experimental site(with an area of 0.93 ha)was selected on flat land to ensure relatively uniform soil properties (Table S1). The site had been one of typical natural alpine Kobresia humilis meadow until 1998,but has since been continuously cultivated with feed oats.Prior to starting the year-round field measurement campaign from 22 April 2013 to 21 April 2014,an unfertilized(UF)treatment was set by randomly establishing four field plots within the site. The remaining part of the site was regarded as the fertilized(F)treatment.Table S1 lists the field management practices of both treatments.Also,four field plots,each with the same size of 5×5 m2as that of the UF treatment, were randomly chosen within the site. The UF plots received the same management practices as the F areas but were free from fertilizer amendment during the experimental campaign to determine the annual direct NO emissions factor.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 fell consecutively within the range from -10°C to 0°C. Accordingly, the spring FTP occurred during the period from 21 February to 21 April 2014. The period from the sowing date to the harvest date,the total remaining time of the full campaign duration,and the full year-round campaign period,are hereinafter referred to as the cropping period(CP),the non-cropping period(NCP),and the annual period(AP),respectively.

NO fluxes from each field plot,which approximated corresponding daily emissions,were measured during the entire campaign using a technique combining the chemiluminescence analysis of NO concentrations with gas sampling by opaque,static chambers.According to the instrument precisions for NO and NO2analysis(0.3 nmol mol–1for both)and the enclosure time(10 min)for gas sampling,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-2h-1,respectively.Due to method-induced underestimation,it should be noted that the NO fluxes measured in this study represented only conservative magnitudes for the investigated ecosystems(e.g.,Zhang et al.2018).Nevertheless,the high sensitivity of the applied method for measuring NO fluxes allowed for investigation of the regulatory effects of soil variables(Zhang et al.2018)and the differences between the F and UF treatments,thus determining the direct emissions factor values(Yao et al.2015).

Air pressure,air temperature and precipitation were observed and provided by HAMERS.Meanwhile,air samples were collected to determine the NO flux,and the corresponding air temperature within the chamber headspace,topsoil(5 cm depth)temperature(Ts)and surface(0–6 cm depth)soil moisture in the water- filled pore space(WFPS)were simultaneously measured.The concentrations of soil(0–10 cm depth)ammonium(NH4+),nitrate(NO3–)and water-extractable organic carbon were observed weekly on one of the days when NO fluxes were measured.Selected soil properties within a depth of 0–20 cm,including the texture,particle fractions,soil organic carbon,total nitrogen,and soil pH,as well as the surface soil(0–6 cm depth)bulk density,were also measured.The aboveground biomass,used to approximate the aboveground net primary productivity(ANPP),and its nitrogen content,were also measured,at harvest.

The annual EFdwas determined as the ratio of the difference between the annual emissions from the F and UN treatments to the annual total rate of fertilizer nitrogen application.Its error was determined from those of the measured NO fluxes.Correlation and regression were adopted as the methods for analyzing the effects of soil variables on NO fluxes.

The SPSS 19.0 software package was used for statistical analysis.The Origin 8.0 software package(Origin Lab Ltd.,Guangzhou,China)was used for plotting the data.The raw experimental data were organized and calculated using the Excel software package of Microsoft Office Standard 2010(? 2010 Microsoft Corporation).

Unless otherwise stated,the standard errors of means for three to five spatial replicates are given to report the results in the text.

For more details on the materials and methods,see Text S1 of the Online Supplementary Material.

3.Results and discussion

3.1.Related variables

The measured soil properties,ANPP,aboveground nitrogen content,and nitrogen uptake are presented in Table S1.The observed meteorological and soil variables are plotted in Figure S1a–d.As Table S2 shows,there were no significant differences in the soil variables between the two treatments.This result accounted for the non-significant difference in the NO fluxes between the F and UF treatments(Figure 1).

3.2.NO fluxes

The measured NO fluxes(Figure S1)were conservative and varied between-0.2 and 28.5 μg N m-2h-1in the F treatment and between-0.2 and 35.7 μg N m-2h-1in the UF treatment,occasionally falling within the negative and positive detection limits.Higher NO fluxes than the annual means(approximately 3 μg N m-2h-1in both treatments)occurred during the two-week period immediately following plow tillage,regardless of whether the operation was accompanied by basal fertilization. These tillage-stimulated fluxes from the F and UF fields during a short period of nearly half a month accumulated to 55 and 57 g N ha-1on average,accounting for approximately 54% and 73% of the cumulative NO emissions during the CP, respectively. The stimulatory effects of plow tillage could be attributed to soil aeration,which improves the oxygen supply for nitrification,whereby NO is produced as a byproduct (e.g., Civerolo and Dickerson 1998;Pinto et al.2004;Skiba,Vandijk,and Ball.2002).In addition,the frequent freeze–thaw alternations occurring during the spring FTP also stimulated definite NO fluxes in both field treatments, which lasted longer than the fluxes stimulated by urea top-dressing (Figure S1f).This freeze–thaw effect could be attributed to the enhanced availability of nitrogen substrates as the NO precursors because of the releases of NH4+and/or organic nitrogen compounds from dead microbes.The nitrogen releases result from the physical damage of microbial cells during the frequent alternations between liquid water and solid ice phases(e.g.,Matzner and Borken 2008;Wolf et al.2010).

The above results indicate that the seasonal variation patterns of NO fluxes from alpine cultivated land for feedoat production are jointly governed by the stimulated fluxes due to plow tillage,fertilization,and freeze–thaw alternation.

3.3.Effects of soil variables on NO fluxes

Figure 1.Conservative nitric oxide emissions from fertilized and unfertilized alpine ecosystems cultivated with feed oats and the contributions of different periods to the annual totals.The values within the bars are the cumulative emissions of the respective period.The error is the standard error of the mean for four spatial replicates.

The NO fluxes typically correlated significantly with Ts(Table S3).The correlations can be further described by the significant exponential dependences on Ts,in which Tsalone accounted for the variance in the NO fluxes by 55%–67%during the NCP,by27%–45%during the CP,and 31%–42%during the AP(Equations(1)–(2),(6),(9)–(10),(14),(17)–(18),and(20)in Table S4).

The NO fluxes also typically correlated positively with NO3–concentrations,significantly in the F treatment during the CP and AP,and in the F plus UF treatments(i.e.,F+UF)during the NCP,CP,and AP,but non-significantly in the other cases(Table S3).These results indicate that,like NO3–,the measured NO was mainly produced by nitrification.This explanation is supported by the too low soil moisture,i.e.,<55%WFPS(Figure S1b,Table S2),for denitrification (e.g.,Davidson,Rogers,and Whitman 1991).Consequently,in comparison with Tsalone,Tsand NO3–concentrations jointly accounted for more variance in the NO fluxes(by 52%–62%versus 27%–45%during the CP and AP)(Equations(2)–(3),(6)–(7),(10)–(11),and(14)–(15)in Table S4).

The NO fluxes correlated positively with NH4+concentrations(albeit not statistically significant)in the F and UF treatments during the CP(Table S3).The correlations hint that heterotrophic nitrification with organic nitrogen substrates,instead of NH4+,might have substantially accounted for the NO emissions(e.g.,Papen et al.1989)in the F and UF treatments during the NCP.

The NO fluxes during the CP correlated negatively with WFPS(but significantly only in F+UF).This result indicates the inhibitory effect of relatively higher soil moisture,which is unfavorable for NO production via nitrification but favorable for NO consumption via denitrification(e.g.,Butterbach-Bahl et al.2013).This explanation is supported by the significantly lower NO3–concentrations in the UF treatment during the CP than during the NCP(Table S2).

The above results imply that soil temperature,followed by NO3–and/or NH4+levels and soil moisture,is the primary factor governing the NO fluxes from cultivated alpine ecosystems with calcareous soils.

3.4.Soil variable–based predictability of NO fluxes

As Equations(5),(8),(13),(16),and(22)in Table S4 display,the five soil variables jointly accounted for the variance in the NO fluxes by up to 72%–76%.In particular,all the selected soil variables,excluding and including soil moisture,explained 67%and 72%of the variance in the AP fluxes across both treatments,respectively(Equations(21)–(22)in Table S4).This finding suggests that temporally and/or spatially distributed NO fluxes across alpine cultivated land with calcareous soil may be easily predicted,providing temporal and spatial data on four or five of the soil variables are available.Before Equation(21)or(22)in Table S4 is applied for accurate prediction,however,the currently estimated parameters relying on conservative NO fluxes still need to be corrected.The parameter corrections require experimental comparison between the observational methods used in this study and other approaches that can be more accurate for measuring NO fluxes(e.g.,dynamic chambers).

3.5.Sensitivity of NO fluxes to temperature change

When significant positive correlations between the concentrations of NH4+and NO3–occurred,e.g.,with correlation coefficients of 0.79 and 0.92 in the F and UF treatments during the CP,respectively(Table S3),there were significant dependences of NO fluxes jointly on NH4+concentrations and Ts(Equations(4)and(12)in Table S4).The regressions to describe the dependences yielded a three-to five-fold greater(p<0.05)temperature response coefficient(Q10)despite comparable coefficients of determination(r2)with those regressions of NO fluxes jointly against NO3–concentrations and Ts(Equations(3)and(11)in Table S4).These results indicate greater temperature sensitivity of the NH4+-initiated nitrification that,along with other NO production processes,substantially accounted for the NO fluxes from both the F and UF treatments during the CP.

The univariate,bivariate or multivariate regressions for the F treatments(Equations(1)–(3)and(5)–(8)in Table S4)resulted in a comparably smaller Q10(approximately 3.6)during the CP and AP than that(5.1)during the NCP.Differently,the regressions for the UF treatments(Equations(9)–(11)and(13)–(16)in Table S4)yielded a significantly greater Q10(p<0.01)than those of the F treatment during the CP or AP(approximately 4.9 versus 3.6),but was not significantly different from those of both treatments during the NCP(4.8–5.1).These results imply that enhanced nitrogen addition to cultivated alpine lands for feed-crop production can significantly reduce the sensitivity of NO emissions to temperature changes,e.g.,due to global warming.The results also suggest that even a short-term halt to nitrogen fertilizer addition(like in the UF case)can greatly increase the temperature sensitivity of NO emissions.This inference means that reducing nitrogen addition to cultivated alpine lands subject to a warming climate may not lead to reduced NO emissions.

3.6.Annual NO emissions

The measured NO fluxes(Figure S1f)amounted to a conservative annual emission of 0.191–0.205 kg N ha–1yr–1on average,showing no significant difference between the F and UF treatments(Figure 1).These annual NO emissions of both treatments were at the level of the lower bound of those(0.2-23 kg N ha–1yr–1)reported by previous studies on cultivated uplands in various sites around the world.They were also typically lower than those of(i)fertilized(0.7–5.7 kg N ha–1yr–1)and short-term unfertilized(0.26–0.60 kg N ha–1yr–1)uplands with calcareous soils,(ii)short term unfertilized vegetable fields and tea gardens with non-calcareous soils(0.33–2.8 kg N ha–1yr–1),and(iii)fertilized vegetable fields and tea gardens with non-calcareous soils(6.6–47.1 kg N ha–1yr–1)(see Table S5 for the means and standard deviations of the previously observed data and their original literature). This finding indicates that the area-scaled NO emission intensities of alpine cultivated lands originating from alpine meadows for the long-term production of feed crops like oats(regardless of whether conventional fertilization practices are used or they are unfertilized in the short term)are clearly much lower than those of non-alpine uplands.The much weaker NO emissions from alpine cultivated lands are primarily attributable to the lower soil temperature.Such an explanation is supported by the significant exponential dependence of NO fluxes on topsoil temperature(Table S4).

3.7.Contributions of special periods to annual NO emissions

Of the annual cumulative NO emissions,the spring FTP,NCP and CP accounted for approximately 17%,51%,and 49%in the F treatment,and approximately 25%,58%,and 42% in the UF treatment,respectively(Figure 1).Due to the significantly stimulated NO fluxes(Figure S1f),the spring FTP accounted for 33%±7%and 43%±2%of the cumulative emissions in the F and UF treatments during the NCP,respectively.There was no significant difference in these contributions between the F and UF treatments.This result implies that shortterm implementation of no nitrogen fertilizer amendment may not modify the seasonal distribution of NO emissions from cultivated alpine lands.However,this implication still requires further confirmation with multi-year field observations that can cover the inherently large interannual variability in precipitation.

Like the present work,a few previous studies on NO emissions from temperate terrestrial ecosystems have shown significant contributions of winter or FTPs(Katayanagi and Hatano 2012;Mukumbuta et al.2017),while other studies have not(e.g.,Filippa et al.2009;Zhang et al.2018).Mukumbuta et al.(2017)reported that the FTPs contributed 1%–32%of the annual NO emissions from grasslands in Japan and adjacent cultivated lands.Zhang et al.(2018)reported marginally low NO fluxes during the NCP at two typical alpine meadows with soil pH below neutral and showed that the FTPs accounted for only approximately 3%of the annual NO emissions.Clearly,the magnitudes of the spring FTP contributions to annual NO emissions from the F and UF treatments fell within these previously reported ranges for temperate or alpine ecosystems(Martin et al.1998;Mukumbuta et al.2017;Zhang et al.2018).

Whether an FTP significantly contributed to annual NO emissions may be related to soil moisture,soil acidity and nitrogen fertilization.Relatively higher soil moisture during an FTPmay reduce its contribution as the NO production in nitrification may be inhibited while consumption of this gas by denitrification may be stimulated(e.g.,Davidson, Rogers,and Whitman 1991).A calcareous soil with pH above neutral may greatly enhance an FTP contribution compared to soils with pH below neutral.Such an effect of soil acidity is shown by the different contributions between the wintergrazed alpine Kobresia humilis meadow, as the origin of the cultivated land where the F and UF field plots were situated(36%±5%(unpublished data)),and the case of wintergrazed alpine Kobresia humilis meadows(approximately 3%)reported by Zhang et al.(2018).Nitrogen fertilization seemed to reduce the FTP contribution,as shown by the different FTP contributions in the F treatment, UF treatment and the adjacent meadow grassland(17%±3%,25%±1%and 36%±5%,respectively).

Martin et al.(1998)reported that the winter season accounted for approximately 25%of the annual NO emissions from temperate grasslands in North America.In typical winter-grazed alpine Kobresia humilis meadows with soil pH below neutral,the NCP accounted for 17%±4%of the annual NO emissions(Zhang et al.2018).Compared to the case of Zhang and his colleagues,the NCP contributions to the annual NO emissions in the F and UF treatments and the adjacent grassland meadow,being 51%±5%,58%±2%and 48%±8%,respectively,were significantly higher.These greater contributions might be mainly attributable to the significantly higher(p< 0.05)soil pH,being 8.3±0.2(Table S1)versus 6.68±0.03(adapted from Zhang et al.(2018)).

3.8.Direct NO emissions factor

The absence of a significant difference in the annual cumulative NO emissions between the F and UF treatments(Figure 1)resulted in a marginally low EFdof 0.021%±0.021%.The EFdwas at the level of the lower bound of those(0.01%-0.36%)for cultivated temperate grasslands in Japan.It was typically lower in comparison with(i)those(0.60%–1.5%)of cultivated savannas,(ii)the previously reported global mean(0.5%–0.7%)of cultivated uplands,(iii)the national mean(approximately 0.67%)of non-alpine uplands in China,(iv)those(0.08%–0.78%,with a mean 0.30%)of non-alpine uplands with calcareous soils,and(v)those(0.33%–2.80%,with a mean of 0.73%)of vegetable fields and tea gardens with non-calcareous soils in non-alpine regions(see Table S5 for the literature of the original data sources).

The marginally low EFdcould be mainly attributed to the low moisture content(below 30%WFPS)during the 10-d period immediately following the basal addition of nitrogen fertilizer(diammonium hydrogen phosphate plus urea)at a rate of 41 kg N ha–1(Figure S1b).The moisture was too low for denitrification,which in turn requires a soil moisture content of greater than 55% WFPS(Davidson,Rogers,and Whitman 1991),thus eliminating the NO contributed by this process.This explanation means that whether or not the EFdvalues obtained in non-alpine regions are applicable in cultivated alpine ecosystems may depend at least partly on the soil water status.Based on this explanation,one can further hypothesize that there might be significantly greater EFdvalues of NO than that of the present study as long as the soil moisture content is high enough(but still unsaturated)to activate nitrifier/denitrifier denitrification during the period immediately following the basal application of urea and/or ammonium-based fertilizers.This hypothesis is proposed because relatively high but unsaturated soil moisture together with relatively low temperature at the beginning of the alpine cropping season is in principle favorable for the formation of NO or nitrous oxide as the byproduct of nitrification,or the production of either gas as an intermediate of the denitrification mediated by denitrifiers and/or nitrifiers (e.g.,Butterbach-Bahl et al.2013;Poth and Focht 1985).However,further field studies are needed to prove this hypothesis.

4.Conclusions

For alpine calcareous-soil ecosystems long-term cultivated with feed oats,a single event of plow tillage to initiate the cropping season can significantly stimulate NO emissions,thus accounting for the cumulative releases during the annual period by more than a quarter,or those during the cropping season by more than a half.Daily NO fluxes are regulated primarily by temperature,secondarily by soil nitrate and/or ammonium levels,and thirdly by soil moisture.It is interesting that bivariate dependences of NO fluxes against ammonium concentrations and temperature occur during the cropping season,yielding a much greater temperature sensitivity coefficient(Q10)than that resulting from univariate dependences against temperature alone or multivariate dependences jointly against temperature and other variables.Nitrogen fertilization practices especially reduce the Q10of NO fluxes during the cropping season.Temporally and/or spatially distributed NO fluxes from cultivated alpine ecosystems,regardless of nitrogen addition levels,may be easily predicted by the soil temperature,concentrations of ammonium,nitrate,and water-extractable organic carbon,providing temporal and spatial data on these variables are available.The non-cropping period accounts for more than a half of annual NO emissions from a cultivated alpine ecosystem.The EFdin cultivated alpine ecosystems are at a marginally low level whenever the soil moisture content during the period following the basal application of urea or ammonium is relatively low.Accordingly,one can hypothesize that there significantly greater EFdvalues than that in this study may occur,as long as the soil moisture content during the period following the basal application of urea-and/or ammonium based nitrogen fertilizer is high enough to activate nitrifier/denitrifier denitrification.However,further field studies are needed to prove this hypothesis.

Acknowledgments

The authors thank Shenghui HAN,Yan LIU,Siqi LI,Han ZHANG,Zhisheng YAO,and Ping LI from the Institute of Atmospheric Physics,Chinese Academy of Sciences,and Le LIU and Xiaowei SHEN for their substantial help with the field and laboratory work in this study.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This study was jointly financed by the Ministry of Science and Technology of China(Grant No.2016YFA0602303)and the National Natural Science Foundation of China(Grant Nos.41775141,41375152,and 41603075).