Introduction
Land use change, a key global change phenomenon, greatly affects ecosystem structure and functioning, particularly, alterations in vegetation types and soil properties induced by land-use profoundly alter soil carbon (C), nitrogen (N) dynamics and microbial activities (Fu et al., 2015; Mbuthia et al., 2015; Rilling et al., 2019; Song et al., 2012; Yue et al., 2020). Although the impacts of land use change on soil C and N pools, and microbial community have been well demonstrated (Cheng et al., 2013; Deng et al., 2016; Moscatelli et al., 2018), the effects of land use change on N-hydrolyzing enzyme activity and stoichiometry remain controversial (Allison et al., 2008; He et al., 2020; Zhang et al., 2019b). For instance, some studies reported conversion of cropland to woodland enhanced N-hydrolyzing enzyme activities and C: N enzyme ratio (Cenini et al., 2016; Feng et al., 2018). Other studies showed that land use change had negative or no effect on N-hydrolyzing enzyme activities or soil enzyme stoichiometry (DeForest and Moorhead, 2020; Fichtner et al., 2014). These inconsistent results can be attributed to different land use management practice and microbial nutrient demand (Sinsabaugh et al., 2016; Wang et al., 2012). Thus, more knowledge on soil N-hydrolyzing enzyme activities in response to different types of land use change is crucial for predictions of N cycling under future global change scenarios.
Soil N-hydrolyzing enzyme including β-1,4-N-acetylglucosaminidase (NAG) and leucine aminopeptidase (LAP) can serve as indicators of energy N demand (Schimel et al., 2017), which catalyze terminal reactions to depolymerize organic substrates for assimilation of N (Sinsabaugh et al., 2009), and are strongly impacted by changes of soil properties under land use change (Acosta-Martinez et al., 2007; He et al., 2020; Sinsabaugh et al., 2008). For example, conversion of cropland to woodland usually tends to increase soil N-hydrolyzing enzyme activities due to increase in soil organic N content as soil organic matter is the substrate of enzymatic reaction (Raiesi and Beheshti, 2014), whereas cultivation decreases the soil N-hydrolyzing enzyme activities due to N fertilizations (Zheng et al., 2020a). Meanwhile, some studies reported that temperature was a key factor explaining the variation in soil N-hydrolyzing enzyme activities (Zhou et al., 2020) with a positive (Waring et al., 2014) and negative (Peng and Wang, 2016; Xu et al., 2017) effects on it. While positive relationship between soil N-hydrolyzing enzyme activities and soil moisture was also be found in some studies (He et al., 2020; Xu et al., 2017). Soil N-hydrolyzing enzyme activity was also affected by soil pH and soil moisture (Fu et al., 2017; Pinsonneault et al., 2016). The changes in soil pH were accompanied by changes in vegetation type which induced positive relationship between soil pH and N-hydrolyzing enzyme activities (He et al., 2020). Despite the importance of the above factors in regulating soil N-hydrolyzing enzyme activities, soil N-hydrolyzing enzyme activities driven by multiple controls under use change are not well examined.
Soil extracellular enzyme stoichiometry (EES) reflects the energy and nutrient controls on microbial metabolism, indicating how shifts in substance and energy allocation alter the according nutrient demand (Cui et al., 2018; Guo et al., 2019; Sinsabaugh et al., 2012; Yang et al., 2020). It has been suggested that ecosystem C: N: phosphorus (P) enzyme stoichiometry greatly varies with land-use types (Hartman and Richardson, 2013; Li et al., 2012). Variations in soil enzyme stoichiometry are consistent with the patterns of microbial nutrient availability under land use change (Sinsabaugh et al., 2008, Waring et al., 2014, Xu et al., 2017). It has been reported that afforestation usually decreases soil enzyme C: N ratio as higher soil C: N ratio induced N limitation for microbial (Feng et al., 2018). But a recent study showed that differences in enzyme stoichiometry between vegetation types were weakly related to the microbial nutrient status (He et al., 2020). Additionally, previous studies have found soil enzyme C: P ratio was inversely related to mean annual temperature (MAT) and precipitation (MAP), while the soil enzyme C: N ratio was positively related to MAP at a global scale (Sinsabaugh et al., 2008). While Cui et al. (2018) reported that plant traits were more important than soil physical and chemical properties in determining EES. Thus, evaluating enzyme stoichiometry patterns and their drivers could improve our understanding of N, P limitations in response to land use change.
The Danjiangkou Reservoir area is a water source for the Middle Route of China’s South-to-North Water Transfer Project. In order to protect water quality of the reservoir and restore riparian ecosystem function, large areas of afforestation have been conducted to reduce soil erosion, water pollution (Zhu et al., 2010). Our previous studies reported that afforestation greatly impacted soil C and N pools, C:N ratios and microbial activities (Cheng et al., 2013; Deng et al., 2016). But study on N-hydrolyzing enzyme activities can be beneficial for understanding the interactions of soil -plant -microorganism for N cycling in this region. Thus, we hypothesized that: (1) afforestation would increase soil N-hydrolyzing enzyme activities primarily due to increased litter inputs (Zhou et al., 2020; Feng et al., 2018), while cultivation (i.e. cropland) would decrease soil N-hydrolyzing enzyme activities possibly due the addition of N fertilizer (Carlos et al., 2021; Zheng et al., 2020b); (2) microbial nutrient acquisition in afforested soils would be limited by N or/and P rather than by C likely due to the nutrients (N or/and P) competition between plants and microbes (Zheng et al., 2020a). To test these hypotheses, we investigated seasonal patterns of NAG and LAP enzyme activities at the top (0-10cm) and deep (10-30cm) under land use change (i.e., woodland, shrubland, cropland and adjacent uncultivated fields). We also examined soil properties including soil moisture, temperature, pH and soil organic C and N (SOC and SON), labile N (LN) and recalcitrant N (RN).