Responses Of Ecosystem Carbon Fluxes To Different Nitrogen Levels And Their Regulatory Factors In An Alpine Steppe

Human activities have led to a significant increase in reactive nitrogen(N)entering terrestrial ecosystems,and the large N inputs have profound impacts on regional and global carbon cycles.Vegetation productivity,soil respiration,and net ecosystem carbon dioxide(CO2)exchange are three key processes in the terrestrial carbon cycle.Productivity determines the total carbon input of the global ecosystem;soil respiration mainly includes root autotrophic respiration,soil animal and microbial heterotrophic respiration;and net ecosystem CO2 exchange is the balance between gross ecosystem primary productivity and ecosystem respiration,and is a direct measure of the net exchange of carbon between terrestrial ecosystems and the atmosphere.Although scholars have conducted numerous studies on how N addition affects key processes of the carbon cycle,the response patterns of the above three carbon flux processes to different N levels and the regulating factors are not yet fully understood.In this study,we investigated the response patterns of carbon flux-related processes and their regulatory mechanisms in a multi-level(0,1,2,4,8,16,24,32 g N m-2 year-1),long-term(2013-2020)N addition manipulation experiment platform in a Tibetan alpine steppe.The main results obtained are as follows:(1)By investigating the characteristics of root production,turnover rate and standing crop at different N levels and analyzing the regulatory effects of soil physicochemical factors and root carbon and N metabolic indicators on the three root processes,we found that root production and standing crop decreased linearly or exponentially with increasing N application.Compared with the control,root production and standing crop in the 16 g N m-2 year-1 treatment decreased on average by 43.0%and 45.7%in 2 years,respectively.Root turnover rate showed an increasing and then decreasing trend,peaking at 2 and 4 g N m-2 year-1 treatments in 2015 and 2016,respectively.The linear mixed-effects model results showed that root starch content was the main factor regulating root production and turnover rate,explaining 21.7%and 25.4%of the variation in both,respectively,while root standing crop was influenced by root protein content,which explained 20.8%.N enrichment showed inhibitory effects on root production and standing crop,low N promoted and high N inhibited root turnover rate,and root carbon and N metabolism indicators were the main factors regulating root dynamics in response to N addition.(2)Using long-term observations of soil respiration and its components from eight consecutive years of N addition experiments and integrating the literature of 171 experiments with more than three N levels worldwide,we found that both soil respiration and autotrophic respiration showed an increasing and then decreasing response pattern with increasing N application from 2013 to 2020,with the threshold value occurring at 8 g N m-2 year-1;heterotrophic respiration showed a linear decreasing trend along the N gradient.At different N levels,N-induced changes in biotic and abiotic factors changed soil respiration by affecting its autotrophic and heterotrophic components.Autotrophic respiration was mainly influenced by changes in root biomass and plant root-to-shoot ratio in the early stage,and other factors such as physiological metabolic processes of the root system in the later stage.Microbial factors played a certain influence in the early stage,but the regulatory role of plant diversity and environmental factors gradually increased in the middle and late stages.Meanwhile,the concave down curves of soil respiration and autotrophic respiration to continuous N input and the linear decreasing response of heterotrophic respiration were universal in the global scale.(3)By studying the patterns of changes in soil microbial diversity,community composition and network relationships at different N levels,as well as the regulatory effects on heterotrophic respiration,it was found that N addition had no significant effect on the α-diversity index of different communities of soil microorganisms(bacterial,fungi and cercozoa)overall compared to the control,and only at high N addition(16,24,32 g N m-2 year-1),it reduced the Shannon-Weiner index of the cercozoa was reduced only at high N additions(16,24,32 g N m-2 year-1).In contrast,microbial community composition was significantly altered at different N levels,especially in blocks treated with>8 g N m-2 year-1.The intra-domain networks of bacterial,fungi and cercozoa did not change significantly with increasing N addition.The cross-trophic network complexity of cercozoa-fungi,cercozoa-bacterial and fungi-bacterial all increased significantly at N levels up to 24 g N m-2 year-1 and tended to saturate or slightly decrease thereafter.Inter-domain interactions between cercozoa-fungi and fungi-bacterial were the main regulators of heterotrophic respiration changes.(4)By analyzing the patterns of changes in gross ecosystem primary productivity(GPP),ecosystem respiration(ER)and net ecosystem CO2 exchange(NEE)with increasing years of N application under different N levels,and establishing relationships between environmental factors(soil temperature,moisture and pH),nutrient(soil and plant N and P)content,plant species diversity and litter biomass with GPP,ER and NEE,it was found that GPP and NEE increased and then leveled off with the increase of N addition at different N levels from 2013 to 2020,reaching the threshold at 8 g N m-2 year-1;ER showed an increasing and then decreasing trend with increasing N application,with a peak also at 8 g N m-2 year-1.The promotion effect of N addition on both GPP and NEE diminished with increasing N application years,and the decline magnitude increased sharply and then leveled off with increasing N application,reaching a threshold value at 8 g N m-2 year-1.In contrast,the response of ER to different N levels did not change significantly with increasing years of N application.The linear mixed-effects model correlation analysis showed that soil temperature and plant species diversity induced by N addition were the main regulators of the temporal effects of GPP and NEE N application,and the relationships between soil temperature and plant species diversity and the temporal effects of GPP and NEE N application were weak under low N addition,and both were significantly positively correlated with the temporal effects of GPP and NEE N application as the N addition gradually increased,the relationship between the two factors and the temporal effect of GPP and NEE was significantly positive with increasing N addition.In summary,this paper analyzes the response of carbon fluxes to different nitrogen levels in alpine grassland ecosystems on the Tibetan Plateau and their regulatory mechanisms,clarifies the response curves of carbon flux-related processes along the nitrogen gradient,and reveals their relationships with biotic and abiotic factors.The results of these studies can help improve the academic understanding of the carbon cycle characteristics of alpine grasslands on the Tibetan Plateau,and can provide a benchmark for testing and improving model simulation results.