Effects of N addition on soil hydrolytic enzymes
In agreement with our first hypothesis and some previous studies (Wang et al. 2008; Heuck et al. 2018; Jia et al. 2020), N treatment significantly increased soil invertase and cellulase mean activities (Fig.1a, b), indicating that N addition had a positive effect on the process of soil invertase and cellulase participating in soil unstable C metabolism (Buchkowski et al. 2015; Sun et al. 2016). The increase of soil invertase and cellulase activities may be related to soil limited target nutrients, that is, N addition increases the N availability relative to C in the soil (Chen et al. 2017, 2018b). Using the excessive N resources, plant roots and microorganisms can synthesize C-hydrolyzing enzymes (such as invertase and cellulase) to accelerate soil labile C mineralization (Wang et al. 2015; Heuck et al. 2018). Notably, the increase of soil total C:P and N:P ratios after N addition maybe also promote soil invertase and cellulase activities, because we found significant correlation between soil invertase activity with total N:P ratio (Fig.2c), and between soil cellulase activity with C:P ratio (Fig.2d). These results support the premise that soil stoichiometric characteristics plays an important role in the regulation of enzymes activities (Fanin et al. 2015; Wang et al. 2020b). However, we found N treatment significantly increased SOC concentration in our study (Table 2), indicating that the increase of soil invertase and cellulase activities did not reduce the accumulation of soil SOC. The increased of SOC concentration could be results from the N-induced suppression of soil microbial biomass (Table 2) (Chen et al. 2018a; Fang et al. 2019), soil respiration (Fang et al. 2019) and decomposition of litter (Whittinghill et al. 2012; Cenini et al. 2016). In our previous studies in the same region, we reported that N addition significantly suppressed soil heterotrophic respiration (Wei et al. 2020), and decomposition of litter and the degradation of lignin and cellulose (Zhou et al. 2018; Tie et al. 2020a). In addition, we found significant correlation between soil cellulase activities with SOC concentration (Fig.2a), this means that the increase of cellulase activity in soil may be related to the increase of substrate utilization (Pancholy and Rice 1973; Katsalirou et al. 2010). Therefore, we believe that although N addition increased SOC concentration, the soil microorganisms growth may also be limited by the C availability, and the increase of soil C-Hy EAs (such as invertase and cellulase) maybe promote the soil unstable C mineralization to improve the availability of C and thus to feed back the growth of microorganisms.
As expected, we found that N treatment significantly decreased the soil urease mean activity in this study (Fig.1c), which in line with some previous N addition experiments (Ajwa et al. 1999; Kang and Lee 2005; Feng et al. 2020). The inhibition effect of N addition on soil urease activity may be explained by the following possible mechanisms. On the one hand, we found that N addition reduced microbial the biomass and soil pH (Table 2), given that significantly positively correlations between soil urease activity with soil pH (Fig.2f) and MBC concentration (Fig.2g), N addition may lead to a decrease in urease activity via N-induced reduction of microbial biomass and the acidification (Table 2) (Burns et al. 2013; Jia et al. 2020). On the other hand, N addition promotes the soil C and P mineralization can continue to release availability N (Olander and Vitousek 2000; Zhu et al. 2014; Wang et al. 2020b), thus inhibit the soil urease activity. The negative correlation between soil urease activity and total C:P (Fig.2d) and N:P (Fig.2e) ratios supported this explanation. In addition, urease is involved in the hydrolysis of urea-type substrates, and its activity is crucial in soil N mineralization (Saiya-Cork et al. 2002; Enowashu et al. 2009; Hu et al. 2013). The continuous N supply to the soil satisfies the nutrient needs of microorganisms and plants, and the rate of N mineralization was reduced (Zhou et al. 2012; Song et al. 2014), then the lower urease activity was observed in our N treatment. However, although N treatment significantly reduced soil urease mean activity in our experiment (Fig.1c), positive effect and no respond also were observed during the first year (Fig.1c). Which mean that N addition may suppressed the soil urease activity by reducing its production, while the process required a certain time to make the urease activity at a lower level (Lloyd and Sheaffe 1973; Burns et al. 2013; Nannipieri et al. 2018). This maybe well explained that there was no significant negative correlation between soil urease activity and TN concentration (Fig.S1d). Being inconsistent with our findings, Sun et al. (2019) who reported that soil urease activity had no response to N addition in a nearby natural evergreen broad-leaved forest, which may be related to the difference of soil N concentration (Trasar-Cepeda et al. 2008; Shi et al. 2018).
Supporting our first hypothesis, we found that soil acid phosphatase activity increased in N treatment (Fig.1d), indicating that greater availability of N stimulated biotic demands of P (Johnson et al. 2005; Hou et al. 2015). Consistent with our results, studies on various ecosystems showed that N addition can promote soil acid phosphatase activity (Yokoyama et al. 2017; Heuck et al. 2018; Jia et al. 2020; Tie et al. 2020b). In this study, N treatment significantly increased soil total C:P and N:P ratios (Table 2), indicating the increase of soil C and N concentrations led to the lack of P in the soil (Tian et al. 2010; Zechmeister-Boltenstern et al. 2011). We also found that soil acid phosphatase activity was positively correlated with total C:P (Fig.2j) and total N:P (Fig.2k) ratios, meaning that with the increase of soil C and N concentrations or the decrease of soil P concentrations, soil acid phosphatase activity will increase. Therefore, the increase of soil acid phosphatase activity may be the result of excessive N resources being used by microbial and plants for the synthesis of phosphatase to enhancing the release of inorganic P from organic P compounds (Mcgill and Cole 1981; Nasto et al. 2014; Zhang et al. 2018). Moreover, soil TP and AP concentrations decreased with the N addition (Table 2) in our research, suggesting that N addition increased soil P uptake by plants and microorganisms (Wang et al. 2007a; Schleuss et al. 2020), which providing additional evidence for microbial and plants production of phosphatase to obtain P in soil (Marklein and Houlton 2012). All of these results imply that soil microorganisms and plants can alleviate soil P deficiency caused by N addition to a certain extent by regulating acid phosphatase activity (Olander and Vitousek 2000; Treseder and Vitousek 2001; Marklein and Houlton 2012). However, the reason for the significantly negative association between the acid phosphatase activity and MBC concentrations was unclear in our study (Fig.1l). We infer the result may be related to the change of soil microbial community composition caused by N addition (Cusack et al. 2011; Jian et al. 2016). In our previous studies revealed that although N addition reduced soil total bacterial biomass, it increased the proportion of bacterial community (the main producer of soil hydrolase) in the 0-20 cm soil layer (Zhou et al. 2017b).
Effects of P addition soil hydrolytic enzymes
In agreement with our second hypothesis, we found that P treatment significantly decreased soil invertase and cellulase mean activities (Fig.1a, b). This result was consistent with the results found in other subtropical forests, where C-Hy EAs were strongly inhibited by P addition (Zheng et al. 2015; Fang et al. 2019; Wang et al. 2020). The decrease of soil invertase and cellulase activities after P addition may be attributed to the following three possible mechanisms: First, P addition may make microbes down regulate their C investments on phosphatase production (Turner and Wright 2014; Wang et al. 2020a). Second, P addition may increase in plant-derived C influx to soils by increased root biomass (Hector 2006; Brzostek et al. 2013; Zhu et al. 2013), and the enrichment in P could release degradable organic C from Fe/Al oxides into soil solution, thus elevating soil C availability (Mori et al. 2018), thereby reducing soil invertase and cellulase activities. This was also supported by our results that soil invertase and cellulase activities had significant positive correlation with soil total C:P ratio (Fig.2b). Third, P addition may reduce in C-hydrolyzing enzymes abundance in soil. On the one hand, P addition may reduce the gene abundance of enzymes that degrade labile C substrates in the soil and decreased the expression of C-hydrolyzing enzymes (Yao et al. 2018). On the other hand, the changes in microbial composition caused by P addition may also reduce soil C-hydrolyzing enzymes abundance. Previous studies have shown that soil MBC had no significant response to P addition in subtropical broad-leaved forests, but greatly increased the relative abundance of symbiotic fungi (Liu et al. 2012), which may reduce the production of C-hydrolyzing enzymes in the soil, because fungi acquire C from host plants rather than producing C degrading enzymes (Gartner et al. 2012). In addition, in this study, we also found that the addition of P significantly reduced the SOC concentration (Table 2), which may be caused by the addition of P to increase the microbial activity in the soil, resulting in a higher rapid of soil organic matter transformation (Liu et al. 2012; Wang et al. 2020c). Therefore, it is not surprising that SOC concentration decreases even if C-Hy EAs is inhibited under P addition. This result may imply that the increase in soil P availability will have a negative impact on soil C sequestration compared with the positive effects of N addition (Reay et al. 2008; Stiles et al. 2017).
Contrary to our expectation, there was no significant effect of P treatment on soil urease mean activity in this study (Fig.1c), meaning that maybe there was no close relationship between soil N demand and urease activity after P addition (Olander and Vitousek 2000). Consistent with our results, P addition had no significant effect on soil urease and other N-hydrolyzing enzymes activities, as indicated by a recent meta-analyze (Xiao et al. 2018) and some experiments (Wang et al. 2020a; Zhang et al. 2020). We speculated that the neutral effect of P addition on soil urease activity observed in this study may be related to the N status of our forest ecosystem. In our study region, the annual average N deposition (wet) reached 95 kg ha-1 (Xu et al. 2013), the higher N deposition may provide sufficient available N for the forest ecosystem in this region (Zhou et al. 2017a). When there was enough N to meet the needs of plants and microorganisms, the production of microbial N-hydrolyzing enzymes will return to the constituent level (Chróst 1991), and soil N-Hy EAs may not change with P addition. In addition, since N is abundant and short-term P addition may not affect the soil N supply (Zhu et al. 2015), a short-term P addition might prevent us from detecting statistically significant changes in soil urease activity. From this, compared with the strict control of phosphatase activity by N addition, the regulation of N-hydrolyzing enzymes by P addition is much weaker in our experimental forest.
In our study, we found P treatment significantly decreased soil acid phosphatase mean activity (Fig.1d), which was consistent with our second hypothesis and the general pattern in most terrestrial ecosystems that P addition have inhibitory effect on soil phosphatase activities (Marklein and Houlton 2012; Xiao et al. 2018), especially in the tropical/subtropical forest ecosystems (Turner and Joseph Wright 2014; Zheng et al. 2015; Yokoyama et al. 2017). In our study, we also found that P treatment significantly reduced the soil total C:P ratio (Table 2). Compared with the higher soil total C:P ratio, the lower soil total C:P ratio after P addition indicated that increased availability of P in the soil, plant and soil microorganisms growth were not restricted by P (Cleveland and Townsend 2006; Mooshammer et al. 2012), the competition between plants and soil microorganisms for P therefore was weakened, thus reducing the microbial dependence on organic P mineralization as the main source of P (Rodríguez and Fraga 1999; Wang et al. 2007b). That is, when the supply of P is sufficient, microorganisms will reduce the investment in phosphatase and the rate of P mineralization will decrease(Marklein and Houlton 2012; Yokoyama et al. 2017). This was also supported by our results that soil AP and TP concentrations had a very significant negative correlation with soil acid phosphatase activity (Fig.2h, i).
Effects of NP co-addition soil hydrolytic enzymes
In our study, NP treatment significantly increased soil invertase and cellulase mean activities (Fig.1 a-b), which is consistent with our third hypothesis. We also found that NP treatment significantly increased soil pH and MBC (Table 2). Therefore, we speculated that the increase in soil invertase and cellulases activities caused by NP treatment might be the result of the increased demand for availability C by soil biological growth (Cleveland et al. 2002; Li et al. 2015). However, unexpectedly, we found NP treatment did not significantly affect the SOC concentration (Table 2), in our study, possible explanation could be the positive effects of N addition on SOC sequestration counteracted by the negative effects of P addition on SOC sequestration, indicating that the SOC mineralization was limited by soil P availability, and P addition could mitigate the effects of N on soil C sequestration in this system (Bradford et al. 2008; Street et al. 2018). In contrast to our third hypothesis that NP treatment would inhibit soil urease and acid phosphatase activities, we found that NP treatment did not significantly change soil urease and acid phosphatase mean activities in our study (Fig.1c, d). The results showed that that under the combination N and P addition, the addition of P might counteract the inhibition of N availability on soil urease activity and the promotion of acid phosphatase activity in subtropical forest soil. We also found that soil N and P concentrations and their stoichiometric ratios were not significantly affected by NP treatment (Table 2), which may explain the minor response of soil urease and phosphatase activities to NP treatment in this study. Consistent with our results, recent meta-analyses also indicated that NP co-addition significantly increased soil C-Hy EAs but the effect on soil N- and P-Hy EAs were not significantly (Xiao et al. 2018).
Different from the effect of alone N or P addition, the effect of NP co-addition on soil hydrolytic enzymes activities may also be the result of the interaction effect of N and P, because there is a widespread close coupling relationship between N and P in the ecosystem (Peñuelas et al. 2013a, b; Gao et al. 2014). Indeed, our results also showed that the interaction of N and P addition had a significant effect on soil invertase, urease, and acid phosphatase activities (Table 1), the interactions found in this study may be attributed to the opposing responses of alone N and P addition on soil C-, P-, and N-Hy EAs, and further research is needed.