Discussion
Our results demonstrated that soil N- hydrolyzing enzyme activities
(including NAG and LAP), and the specific enzyme activities varied with
land use change and season in the Danjiangkou Reservoir area of central
China (Table 2; Figs. 1-3). Notably, whether conversion of uncultivated
land to the afforested lands (woodland and shrubland) or cropland
enhanced NAG and LAP enzyme activities in this region. With land use
changing, multiple factors including availability of nutrients, soil
microbial attributes and soil properties could cause variation in soil
enzyme activities and EES (Luo et al., 2020; Zhao et al., 2018).
Firstly, greater SOC and SON contents in afforested soils and cropland
soils compared to the uncultivated land (Table 1; Fig. 5) made larger
contribution to increases in NAG and LAP enzyme activities, because SOC
and SON could provide more substrates and energy for enzymatic reactions
(Feng et al., 2018). The significant positive relationships of soil
N-hydrolyzing enzyme activities with SON, SOC (Table 1; Table S1)
further confirmed that the SON and SOC were importance source for
microbes producing hydrolytic enzymes (Wallenstein & Weintraub, 2008;
Yu et al., 2017). Increased N-hydrolyzing enzyme activities in the
afforested soils could also be attributed to larger LN contents as
labile organic matter were preferentially used by microorganisms (Knops
et al., 2002). The tightly positive relationship between LN and
hydrolyzing enzyme activities were found in our Pearson’s correlation
analysis (Table 1). Secondly, the higher soil C: N ratios (i.e., the C:
N ratio of organic soils, C: N ratio of labile pool, C: N ratio of
recalcitrant pool) could possibly lead to higher N-hydrolyzing enzyme
activities as microbial activities was more susceptible to N restriction
(Mendham et al., 2004). This speculation was supported by the
significant positive correlation between soil N-hydrolyzing enzyme
activities and three kinds of C: N ratios across all land types (Table
1). Thirdly, our results showed that two N- hydrolyzing enzyme
activities was negatively correlated with
NH4+-N and
NO3--N (Table 1). This result also
agrees with the results of other researchers, which showed that
increased inorganic N availability could inhibit the synthesis of
N-hydrolyzing enzyme activities (Allison et al., 2007; Stursova et al.,
2006). Additionally, our results showed that change in soil pH under
land use change negatively affected soil N-hydrolyzing enzyme activities
(Table 1 and Table S1), which highlighted the evidence that soil pH was
a strong control on enzyme activities with soil pH values of
approximately 8 (Cenini et al., 2016).
As expect, part of our finding was consistent with our hypothesis 1 that
afforestation would increase soil N-hydrolyzing enzyme activities
compared with uncultivated soils. Whereas, cultivation also increased
soil N-hydrolyzing enzyme activities compared with those in the
uncultivated land soil, which was contrary to part of the hypothesis 1
(Figs. 1-2). This could attribute to the N fertilization could not meet
the nutritional needs of crops and microorganisms in cropland.
Generally, N- hydrolyzing enzyme activities decreased with the soil
depth (Figs. 1-2), primarily due to the decrease in C and N availability
with increasing soil depth (Bowles et al., 2014). Meanwhile, the
seasonal pattern of both NAG and LAP enzyme activities with higher
levels in summer than other seasons could be attributed to higher
microbial biomass in summer (Fig. 2). Globally, temperature has positive
influence on the activities of N-hydrolyzing enzymes (Sinsabaugh et al.,
2008). However, there was no significant relationship between soil
temperature and soil N-hydrolyzing enzyme activities in our study region
(Table 1). Instead, we found the positive relationship between soil
total PLFA biomass and soil NAG and LAP enzyme activities (Table 1), due
to more favorable temperature in summer (Zhang et al., 2016; Zhang et
al., 2019a; Table 1)
Specific enzyme activities increased with both cultivation and
afforestation (Fig. 3), confirming that increased N turnover rate and
enhanced N mineralization via N-hydrolyzing enzyme activities (Wang et
al., 2012). It has been suggested that specific enzyme activities can
decouple the soil enzyme activities from changes in SON contents, which
can serve as an improved indicator of the co-metabolism of microbial
communities (Raiesi and Beheshti 2014; Sinsabaugh et al., 2009). We also
found specific enzyme activities were significantly lower in cropland
compared to afforested soils (Fig. 3), being consistent with previous
results that N fertilization in cropland reduced the activity of NAG and
LAP per unit of soil SON (Cenini, 2016). The addition of inorganic forms
of N to soils could provide N forms available for microorganism to be
assimilated, which could explain the lower N-hydrolyzing enzyme
activities and lower specific enzyme activities in cropland compared to
the afforested lands.
Previous studies indicated that soil ecoenzymatic C: N, C: P and N: P
ratios reflected the metabolic requirements and nutrient availability of
microbes in the environments (Cui et al., 2018; Waring et al., 2014).
The soil ecoenzymatic C: N and C: P ratios were averaged 0.72 and 0.63
in our dataset, respectively (Fig. 4), which was higher than the global
values (0.62 and 0.13) (Sinsabaugh et al., 2008, 2009). This finding
implied that soil microbial growth and C uptake in our study area could
be limited by N and P availability (Xu et al., 2017). Peng and Wang
(2016) also reported higher soil ecoenzymatic N: P ratio in temperate
grasslands than in tropical soils, indicating higher N demand compared
to P demand in temperate grassland. Meanwhile, we found that soil
ecoenzymatic C: N and C: P ratios were higher in the afforested lands
and croplands than in the uncultivated land, indicating that greater
microbial demand for C relative to N and P with afforestation and
cultivation. Higher soil ecoenzymatic C: N and C: P ratios in cropland
land and afforested land could be related to the different nutrient
status of soil microorganism (He et al., 2020). According to resource
allocation strategy, microbes produce additional N-hydrolyzing enzymes
(indicated by low soil ecoenzymatic C: N ratios) in nutrient-limited
environments (Xu et al., 2017). However, the soil ecoenzymatic C: N
ratios increased with increasing soil C: N ratios in this present study
(Table 1). We inferred that the enzymatic acquisition did not match the
resource stoichiometry because SOM with labile and recalcitrant
fractions, possibly only a small labile fraction could be used by
microorganisms (Kamble and Bååth, 2014). This speculation was supported
by the higher RIC and RIN index (i.e. larger recalcitrant fraction) in
afforested soils (Table 1).
In addition, we found that soil ecoenzymatic N: P ratio was
significantly higher in cropland than other land uses (Fig. 4), due to P
fertilizer in cropland. During afforestation, plant debris input in
woodland and shrubland, possibly increased soil N content (Cheng et al.,
2013; Zhang et al., 2016), while soil P was mainly derived from mineral
decomposition and fertilizer. The addition of P fertilizer to farmland
could weaken the P limitation. All ecoenzymatic ratios (ecoenzymatic C:
N ratios, ecoenzymatic C: P ratios and ecoenzymatic N: P ratios)
decreased with depth in afforested soils, suggesting that microbes could
invest more N- and P-hydrolyzing enzymes than C-hydrolyzing enzymes,
especially in deep soils. Notably, soil enzymatic C: N: P hydrolyzing
enzyme activities averaged from 1: 1.06: 1.12 to 1: 2.20: 2.50 in all
land use types (Table 3). The ratios of soil C-, N-, and P-hydrolyzing
enzyme activities converged on 1:1:1 at the global scale,
that defined the boundaries of
microbial responses to changes in resource availability and resource
allocation patterns depending on nutrient demands (Sinsabaugh et al.,
2008). Thus, our results significantly deviated from 1:1:1. It indicated
that soil microbes were co-limited by C and P with the imbalance between
resource supply and production N and P limitation in our study region.