Discussion
The results from our field experiments show that the alpine steppe was a
net source of N2O. N addition significantly increased
N2O emissions (Figure 2). Most terrestrial ecosystems,
especially grassland ecosystems, are widely limited by N (Geng et al.,
2019; Lu et al., 2011). N enrichment increases N available in soil, even
reaching N saturation, and available N directly affects
N2O emissions (Peng et al., 2018). In our experiment, N
addition significantly increased inorganic N in soil (Table 2).
N2O emissions occurred mainly due to the supply of
substrate NO3−-N, independent of
NH4+-N (Figure 4), which indicates
that denitrification may be the predominant pathway of
N2O emissions in this alpine steppe. A possible
explanation for this finding is that the N supply may lead to plants and
microorganisms competing for NH4+-N
(as a substrate for nitrification) in N-limited grassland ecosystems.
Liu et al. (2013) discovered that N input promotes plant N uptake,
especially NH4+-N. In this case,
nitrification might have been inhibited due to lack of substrates. We
also found that changes in abiotic factors such as soil temperature and
pH regulated N2O emissions. Generally, soil N cycling
largely depends on soil temperature in alpine ecosystems. In particular,
warming was found to drive N2O production and emissions
(Griffis et al., 2017). However, rising temperatures negatively affected
N2O emissions in our study (Figure 4). It is possible
that higher temperatures aggravate evapotranspiration and decrease soil
water availability, thereby limiting various microbial N cycling
processes (Shi et al., 2012). Previous studies have also shown that soil
acidification caused by N saturation limits microbial growth, thus
restraining N2O emissions (Oertel et al., 2016;
Treseder, 2008). In contrast, we found that lower pH contributed to
N2O emissions (Figure 4). A possible explanation for
this discrepancy is that even though N addition significantly decreased
soil pH, the soil was still alkaline (Table 2) and therefore microbial
activity was not negatively affected. It is worth noting that plant
biomass is also a key driver of N2O emissions. Soil
labile C via root secretion may accelerate N2O emissions
because denitrification is commonly driven by high available C as a
source of energy (Li et al., 2020). This phenomenon is consistent with
our conclusion that the increase of belowground biomass boosted
N2O emissions (Figure 4).
Changed precipitation regimes also play an important role in modulating
soil N cycling (Chen et al., 2013; Cregger et al., 2014; Lin et al.,
2016). Li et al. (2020) demonstrated that increased precipitation
exacerbated N2O emissions in grassland ecosystems while
reductions in precipitation mitigated N2O emissions. In
this study, however, we observed that altered precipitation patterns did
not affect N2O emissions (Figure 2). On the one hand,
water addition may diminish soil N pools (soil inorganic N) by promoting
plant N uptake and soil leaching, neither of which are conducive to
nitrification and denitrification (Austin et al., 2004; Kruger et al.,
2021; Lin et al., 2016). On the other hand, water reduction (i.e.,
prolonged drought treatment) had little effect on N2O
emissions, possibly because the alpine steppe itself belongs to an arid
grassland ecosystem and is insensitive to drought treatment (Dijkstra et
al., 2013). The interaction between altered precipitation regimes and N
addition did not significantly affect N2O emissions in
our experiment (Figure 2). There are several mechanisms that could
contribute to this finding. Ordinarily, N and water co-limitation is a
typical feature of arid grassland ecosystems (Austin et al., 2004; Lü et
al., 2009). The responses of grassland ecosystems to N deposition are
strongly regulated by precipitation patterns (Harpole et al., 2007).
Increased precipitation, particularly under the background of N
addition, could increase plant access to soil inorganic N resources (Li
et al., 2019), so the effect of N addition on N2O
emissions may be alleviated by water addition. In addition, decreased
precipitation may suppress microbial activity, leading to inefficient N
assimilation, despite the presence of large amounts of N substrates in
the soil (Homyak et al., 2017; Li et al., 2020). Overall, precipitation
changes attenuated N2O flux responses to N addition,
thus mitigating N2O emissions on the QTP.
The community composition and diversity of N cycling microbes are
directly involved in N2O production and emissions.
Microbial functional genes associated with N cycling encode some key
oxidoreductases and are therefore used as genetic markers for nitrifying
and denitrifying microorganisms (Mushinski et al., 2021). The functional
genes of AOA and AOB usually regulate the rate-limiting step (ammonia
oxidation: NH3 → NH2OH) in nitrification
(Hu et al., 2015; Lu et al., 2015). Some studies have indicated that
N2O emissions were promoted by increased abundances of
both AOA and AOB (Brin et al., 2019; Linton et al., 2020). However, we
found that N addition only significantly increased the abundance of AOB
(Figure 3), and the functional genes of AOB rather than those of AOA
dominated the N2O emissions from nitrification (Table
3). Di et al. (2009) also showed that N2O emissions are
driven by AOB and not AOA in N-enriched grassland ecosystems. Previous
investigations demonstrated that AOA and AOB occupy different niches.
AOA and AOB play a dominant role in acidic and alkaline soils,
respectively, and pH is the chief factor for niche separation (Hu et
al., 2015; Tzanakakis et al., 2019). The alkaline conditions in this
study may be more conducive to the activity of AOB, which further
supports our conclusion that AOB controlled the N2O emissions in
nitrification. The key step of denitrification
(NO2− → NO) is generally mediated bynirS - or nirK -encoding nitrite reductase (Butterbach-Bahl
et al., 2013). In this study, N2O emissions were not
related to the abundance of nirS and nirK (Table 3). This
finding can be explained by other environmental factors such as soil
temperature, pH, labile carbon, and oxygen concentration dominating the
underlying ecological process (Li et al., 2020). The nitrous oxide
reductase encoded by nosZ promotes N2O reduction
(N2O → N2), thereby reducing
N2O emissions (Butterbach-Bahl et al., 2013; Hu et al.,
2015). Decreased nosZ abundance is unfavorable to the reduction
of N2O, thus aggravating N2O emissions
(Bowen et al., 2020). We found that N addition decreased nosZabundance to some extent, and N2O flux was negatively
correlated with nosZ . Thus, the lower nosZ abundance may
be responsible for the increased N2O emission in
denitrification. Although changes in nirS and nirK had no
effect on N2O emission, the high ratios of
(nirS+nirK )/nosZ induced N2O emissions
(Table 3). Given that the high ratios of (nirS+nirK )/nosZrepresented a strong N2O emissions capacity (Hu et al.,
2015), the nirS - or nirK -containing denitrifiers cannot be
ignored in future work on N cycling.