This is the accepted version of the article: Wang, Ruzhen; Cao, Yanzhuo; Wang, Hongyi; [et al.]. «Exogenous P compounds differentially interacted with N availability to regulate enzymatic activities in a meadow steppe». European Journal of Soil Science, Vol. 71, issue 4 (July 2020), p. 667-680. DOI 10.1111/ejss.12906 This version is avaible at https://ddd.uab.cat/record/232204 under the terms of the license 1 Exogenous P compounds differentially interacted with N availability to regulate 2 enzymatic activities in a meadow steppe 3 4 Ruzhen Wanga,1,*,Yanzhuo Caoa,b,1, Hongyi Wanga,c, Feike A. Dijkstrad, Jinglun Jianga, 5 Ruonan Zhaoa, Wang Maa, Tianpeng Lia, Maxim Dorodnikove, Zhengwen Wanga, Jordi 6 Sardansf,g, Josep Peñuelasf,g 7 a 8 Erguna Forest-Steppe Ecotone Ecosystem Research Station, Institute of Applied 9 Ecology, Chinese Academy of Sciences, Shenyang 110016, China b 10 Key Laboratory of Regional Environment and Eco-Remediation, College of 11 Environment, Shenyang University, Shenyang 110044, China c 12 Heilongjiang Bayi Agricultural University, Daqing 163319, China d 13 Sydney Institute of Agriculture, School of Life and Environmental Sciences, The 14 University of Sydney, Sydney, NSW 2006, Australia e 15 Department of Soil Science of Temperate Ecosystems, Georg-August University of 16 Göttingen, Büsgenweg 2, 37077 Göttingen, Germany f 17 CSIC, Global Ecology Unit CREAF-CSIC-UAB, 08913 Bellaterra, Catalonia, Spain g 18 CREAF, 08913 Cerdanyola del Vallès, Catalonia, Spain 1 19 Authors contributed equally to this work. 20 * 21 Corresponding author: Ruzhen Wang, Tel: +86 24 83970603; Fax: +86 24 83970300 22 E-mail address: ruzhenwang@iae.ac.cn 1 23 Summary 24 Increased inputs of ecosystem nitrogen (N) and phosphorus (P) may affect the activity 25 of soil enzymes that play essential roles in the metabolization of carbon (C), N and P 26 for microbial growth. However, the associations between soil enzymatic activities and 27 N and P availability remain poorly understood. We conducted a study in a meadow 28 steppe to elucidate the effects of the addition of N, as ammonium nitrate (NH4NO3), 29 and two forms of P with contrasting solubility, comprising monopotassium phosphate 30 (KH2PO4) that is more soluble than triple superphosphate (Ca(H2PO4)2, on activity of 31 β-glucosidase (BG), N-acetyl-glucosaminidase (NAG) and acid and alkaline 32 phosphomonoesterases (PMEs). In general, there was a positive effect of N on BG, 33 NAG and alkaline PME activity as a result of enhanced soil N availability, plant- 34 microbe nutrient competition and plant P uptake. Addition of KH2PO4 increased activity 35 of BG, NAG and alkaline PME, but had no impact on acid PME activity. Addition of 36 Ca(H2PO4)2 increased NAG activity, but only increased activity of BG and alkaline 37 PME with the addition of N. Concentration of soil available P and microbial biomass P 38 increased with added P, particularly KH2PO4. These results provide the first evidence 39 for the N- and P-mediated stimulation of microbial activity depending on the chemical 40 form of added P in this ecosystem. Relationships between activity of BG and NAG, and 41 between that of NAG and PME were allometric, indicating disproportionate changes in 42 activity of these soil enzymes. This further suggests shifts in microbial acquisition of C, 43 N and P along with increases in availability of N and P that may potentially affect plant 44 productivity. We conclude that scenarios of global environmental change, in which 2 45 ecosystem availability of N and P are affected, may result in variable activity responses 46 among soil enzymes, while the chemical form of P input should be considered as an 47 important factor influencing meadow steppe grassland ecosystem function. 48 49 Keywords enzymatic stoichiometry, extracellular enzymatic activity, microbial 50 biomass phosphorus, nitrogen availability, phosphorus fertilization 51 52 Highlights 53  Chemical N and P increased enzyme activity but effects varied with P form 54 and rate. 55  N addition promoted soil enzyme activity through enhanced plant-microbe 56 interactions. 57  P and N availability resulted in variable activity responses among soil enzymes. 58  Enzymatic stoichiometry showed varying microbial responses in C-, N- and P- 59 acquisition. 60 3 61 Introduction 62 Current inputs of nitrogen (N) to ecosystems are 2- to 3-fold greater than levels prior to 63 the green revolution and are >4-fold greater than those of phosphorus (P) (Peñuelas et 64 al., 2013; Wang et al., 2018). Although global anthropogenic N inputs have steadily 65 increased from 120-150 Mt y-1 in the 1980s to 165-250 Mt y-1 in the 2000s (Peñuelas 66 et al., 2012) and are largely derived from crops that fix N2, industrial fertilizers and 67 emissions from fossil fuels, global anthropogenic inputs of P, which mostly stem from 68 fertilizer use, have remained relatively stable. Thus, anthropogenic inputs of N and P 69 have become increasingly unbalanced, with N:P ratios that are often much greater than 70 those for terrestrial plants (Peñuelas et al., 2012, 2013). Changes in N and P cycles 71 influence ecosystem stability and functions, such as primary productivity, plant-litter 72 decomposition, nutrient release and C balance, particularly in temperate and boreal 73 (limited by N) and tropical (limited by P) regions (Peñuelas et al., 2013; Fernández- 74 Martínez et al., 2014; Jing et al., 2016; Niu et al., 2016; Chen et al., 2017). 75 Soil enzymes play a key role in the decomposition of soil organic matter and 76 recycling of soil nutrients for plant and microbial growth (Shukla & Varma 2011; 77 Trivedi et al., 2016). For example, β-glucosidase (BG) enzymes, which hydrolyze 78 cellulose and other β-linked glucans into glucose, and N-acetyl-glucosaminidase (NAG) 79 enzymes, which hydrolyze chitin and other β-linked aminopolysaccharides into 80 glucosamine, are commonly used indicators of microbial C and N acquisition, 81 respectively (Carreiro et al., 2000; Sinsabaugh et al., 2014). Acid and alkaline 82 phosphomonoesterases (PMEs), required to hydrolyze phosphate from phospholipids 4 83 and phosphosaccharides are used as indicators of microbial P acquisition (Sinsabaugh 84 et al., 2014; Jian et al., 2016). β-glucosidaseBG, NAG and acid/alkaline PME catalyze 85 terminal and rate-limiting reactions to produce C, N and P products that are assimilable 86 by microbes, so their activities represent microbial C, N and P demand (Tabatabai, 1994; 87 Sinsabaugh et al., 2014). As a result, these four enzymes have been used in studies to 88 improve understanding of C, N and P cycling in soils (Sinsabaugh et al., 2009, 2014; 89 Waring et al., 2014; Cenini et al., 2015). Soil C-cycling enzymes regulate the activity 90 of N- and P-cycling enzymes via influencing microbial C availability and 91 consequentially enzymatic activity, so activities of these enzymes are often tightly 92 coupled with stoichiometric relationships (Waring et al., 2014). Activities of soil 93 enzymes have been shown to be positively correlated with plant productivity (Margalef 94 et al., 2017; Sterkenburg et al., 2018), plant nutrient demand (Sardans et al., 2007) and 95 soil C:N:P stoichiometry of an ecosystem (Sinsabaugh et al., 2009), and may be 96 affected by anthropogenic mediated changes in ecosystem availability of N and P. 97 However, the magnitude and direction of single and combined effects of N and P inputs 98 remain uncertain. 99 Effects of N addition on soil enzymatic activities in grassland ecosystems have 100 been widely studied (summarized in Wang et al. 2014), and) and have been shown to 101 vary. For example, positive, negative and neutral effects of soil N availability on C- and 102 P-cycling enzyme activities have been reported (refer to Wang et al., 2014), indicating 103 that enzyme activity may be mediated by other soil physicochemical properties, such 104 as soil pH, moisture and P availability (Sardans et al., 2007; Sinsabaugh et al., 2008; 5 105 Waring et al., 2014; Mori et al., 2018). Indeed, N-induced soil acidification has been 106 found to decrease BG and PME activities by inhibiting microbial growth, but higher 107 levels of N availability led to reduced limitation of microbial N (Yang et al., 2017). 108 Effects of P addition, particularly those related to chemical form of P, on enzymatic 109 activities (Table 1) and potential ecosystem responses in grasslands are less clear than 110 those of N. For example, the addition of the relatively soluble monosodium phosphate 111 (NaH2PO4) increased plant productivity on average by 22% in 98 grassland soils across 112 North America (Craine & Jackson, 2010), while the less soluble triple superphosphate 113 (Ca(H2PO4)2) elicited positive (+59%) and neutral effects in South African grasslands 114 (Craine et al., 2008), and. And additions of NaH2PO4 and Ca(H2PO4)2 decreased 115 (Phoenix et al., 2003) and increased (Tian et al., 2016) PME activity in natural 116 calcareous grasslands, respectively. 117 These contrasting effects of different chemical forms of P on enzyme activity may 118 be due to differences in soil pH, P-absorption capacity, time since application and their 119 varying use efficiencies by plants and soil microorganisms related to P compound 120 chemistry (Mori et al., 2018). For example, KH2PO4 addition was found to increase BG, 121 NAG and acid PME activities in both a savannah and a semi-natural grassland (Mganga 122 et al., 2015), while superphosphate application had no impact on the activity of the three 123 enzymes in an alpine grassland (Jing et al., 2016). However, no comparison has been 124 made for the differential effects of soluble and less soluble P forms on enzyme activities 125 in the same grassland ecosystem with the same environmental conditions and 126 fertilization history. Pre-input levels of ecosystem N and P are essential factors that 6 127 influence microbial activity in response to P inputs (Waring et al., 2014; Tian et al., 128 2016; Margalef et al., 2017), as demonstrated by increased PME activity in a P-poor 129 steppe in response to P addition, but decreased activity in a relatively P-rich old-field 130 grassland (Tian et al., 2016). Although little is known about the combined effects of 131 inputs of N and chemical forms of P on C-, N- and P-cycling enzyme activities, 132 microbial economic theory suggests that higher levels of P availability suppress P- 133 cycling enzyme activity, but promote C- and N-cycling enzyme activities (Allison et 134 al., 2010), where these effects would be stronger with more soluble forms of P. 135 The meadow steppe of northeastern China represents one of the dominant types of 136 grassland in Eurasia and plays a vital role in supporting the regional economy, floral 137 diversity and environmental health (Wang & Ba, 2008). This cold meadow steppe is 138 highly sensitive to global climate change and is within the vulnerable ecotone between 139 forest and steppe that receives 1-2 g N m-2 y-1 (Xu et al., 2015). Given increased 140 productivity of the grassland is required to support a growing human population, more 141 efficient P fertilization is urgently needed to prevent the gradual depletion of natural P 142 reserves (Sattari et al., 2016). Therefore, we investigated the effects of combined 143 additions of N and chemical forms of P on ecosystem processes in a meadow steppe 144 field experiment, to test the hypotheses that (1) combined P and N addition would 145 alleviates the decrease of NAG and increase of PME activities but stimulate the increase 146 of BG activity as caused by N addition alone due to increasing microbial C and N 147 requirements with enhanced P inputs; and (2) Ca(H2PO4)2 would hasve less impact on 148 enzyme activities than KH2PO4 where increases in BG and NAG activities and 7 149 decreases in PME activities are more pronounced under the more soluble form of P. 150 151 Materials and methods 152 Study site and experimental design 153 The field experiment was conducted at the Erguna Forest-Steppe Ecotone Research 154 Station, Chinese Academy of Sciences, located at the southern boundary of the Eurasian 155 permafrost region in Inner Mongolia. The climate of this area belongs to the transition 156 zone between cold- and mid-temperate climates, with mean annual temperature and 157 precipitation of -3 °C and 375 mm, respectively. The meadow steppe ecosystem is 158 dominated by Leymus chinensis (Trin.) Tzvel, Stipa baicalensis Roshev and Carex 159 duriuscula C.A.Mey, and soils are classified as Chernozem (IUSS Working Group 160 WRB 2014). The relatively low background inputs of N and P render this an ideal 161 location for the study of ecosystem responses to global environmental change and 162 nutrient management. The bulk soil pH was 6.8 ± 0.07. Elemental analysis showed the 163 bulk soil to have 24.0 ± 0.57 g kg-1 C, 1.8 ± 0.06 g kg-1 N and 0.5 ± 0.02 g kg-1 P. 164 The experiment started in May 2014, and annual applications of fertilizers in May 165 comprised KH2PO4 or Ca(H2PO4)2 applied at 0, 2, 4, 6, 8 and 10 g P m-2 y-1, with 166 (elevated) and without (ambient) N, applied as NH4NO3 at 0 and 10 g N m-2 y-1, 167 arranged as three factors in a randomized block design, with five replicates; blocks were 168 separated by 2-m buffer strips, and the 24 plots (8 × 8 m) within a block were separated 169 by 1-m buffer strips (Figure S1). Fertilizers were applied to the soil surface as pellets. 170 We chose 10 g N m-2 y-1 as it has been suggested to be the upper threshold for affecting 8 171 aboveground productivity, species richness and composition of plant functional groups 172 in Inner Mongolian grasslands (Bai et al., 2010). However, an upper threshold for P has 173 not been clearly established. In this study, we chose two commonly-usedcommonly 174 used P fertilizers of KH2PO4 and Ca(H2PO4)2 to compare their effects on soil enzyme 175 activities. We used KCl in the KH2PO4 plots to ensure similar levels of potassium (K) 176 that were equal to those in the highest P treatment (13.2 g K m-2); CaCl2 was used to 177 maintain a constant annual chloride (Cl) input (12.1 g Cl m-2) with KCl addition. 178 Calcium was not compensated, because of its high natural abundance in the calcareous 179 soils. 180 181 Field sampling 182 Aboveground biomass of all plant material, including litterfall, was harvested from a 1 183 × 1-m quadrat that was placed randomly in each plot in August 2016. Plants were sorted 184 to species and oven-dried with the litterfall biomass at 65 ºC for 48 h, and then weighed. 185 Five samples of soil, which were collected from the 0-10 cm layer using a 5-cm 186 diameter auger in August 2016, were combined to form a single composite sample per 187 plot and then stored in insulated cans at 4 °C during transport to the laboratory. The soil 188 samples were sieved through a 2-mm screen to remove stones and visible roots; samples 189 were subdivided, where one subsample was stored at 4 °C until analysis of microbial 190 parameters, and another was air-dried. 191 Soil pH was determined in a 1:5 (w/v) soil-water suspension using a PHS-3G digital 192 pH meter (Precision and Scientific Instrument Co. Ltd., Shanghai, China). A subsample 9 193 of the air-dried soil was ground using a ball mill (Retsch M400, Haan, Germany) for 194 analysis of soil organic C (SOC), total N (TN) and total P (TP). SOC and TN 195 concentrations were determined using K2Cr2O7 oxidation (Nelson & Sommers, 1982) 196 and the Kjeldahl method (Bremner, 1996), respectively, and soil TP concentration was 197 determined using molybdenum-blue colourimetry following acid digestion of 0.1 g of 198 soil (HNO3, HClO4 and HF). Total P concentration in the three dominant plant species 199 (L. chinensis, S. Baicalensis and C. duriuscula) was determined using molybdenum- 200 blue colorimetry following acid digestion of 0.3 g of plant material (HNO3 and HClO4) 201 (Murphy & Riley, 1962) and P uptake in the species was determined as the product of 202 its biomass and P concentration. Total inorganic N (TIN) concentration was calculated 203 as the sum of NO3--N and NH4+-N concentrations that were determined using a 204 continuous-flow analyzer (AutoAnalyzer III, Bran & Luebbe, Norderstedt, Germany), 205 following extraction from fresh soil using 2 M KCl. 206 Microbial biomass C (MBC) was determined using fumigation-extraction (Vance et 207 al., 1987), where a 15-g subsample of fresh soil was fumigated with chloroform for 24 208 h; another 15-g subsample was not fumigated. Following extraction with 0.5 M K2SO4, 209 the concentration of C was determined using a TOC analyzer (Multi N/C 3100, Analytik 210 Jena, Jena, Germany), and MBC concentration was calculated as the difference between 211 the fumigated and unfumigated samples. To correct for incomplete extraction, an 212 efficiency factor of 0.38, which has previously been used for this grassland, was used 213 to calculate actual MBC concentration (Wang et al., 2015). Concentration of microbial 214 biomass P (MBP) was determined as for MBC, except we used 0.5 M NaHCO3 as the 10 215 extractant (Brookes et al., 1982). Phosphate concentration in the extracts was measured 216 using molybdenum-blue colourimetry with a UV-visible spectrophotometer (UV-1700, 217 Shimadzu, Kyoto, Japan) (Murphy & Riley, 1962). The efficiency factor for MBP (0.39) 218 was determined according to the equation proposed by Bilyera et al. (2018), using SOC 219 (24.5 g kg-1), total P (0.53 g kg-1) and clay content (23.6%). Extractable C in the 220 unfumigated samples was classed as dissolved organic C (DOC), and available (Olsen) 221 P was extracted from 2.5 g of air-dried soil with 50 ml of 0.5 M NaHCO3 (pH 8.5) 222 (Olsen et al., 1954) and then filtered; Olsen P was measured using molybdenum-blue 223 colourimetry. Biomass of arbuscular mycorrhizal fungi (AMF) was estimated from 224 analysis of phospholipid fatty acids that were extracted, fractionated and quantified 225 (after Bossio & Scow, 1998) from frozen soil samples; the extraction mixture comprised 226 CHCl3, methanol and citrate buffer (1:2:0.6). The extracted phospholipids were 227 separated from neutral lipids and glycolipids and then methylated into fatty acid methyl 228 esters via a mild alkaline methanolysis; the fatty acid methyl esters were then analyzed 229 using a gas chromatograph (Agilent 7890A, Wilmington, USA) and identified using a 230 microbial identification system (Microbial ID. Inc., Newark, DE, USA). We used fatty 231 acid 16:1ω5 as a biomarker for AMF (Zhang et al., 2015). 232 233 Soil enzymatic activity 234 Maximum potential activities of BG, NAG and acid/alkaline PME required to catalyze 235 specific reactions were determined at their respective optimal pHs and temperatures 236 from fresh soil samples to allow comparison with other studies. The incubation 11 237 temperature was not adjusted to the mean annual temperature (-3 °C) that may have 238 been a rate-limiting factor and obscured any treatment effects (Nannipieri et al., 2018). 239 For BG activity, 1 g of soil was mixed with 0.25 ml of toluene, 4 ml of modified 240 universal buffer (comprising 0.1 M tris(hydroxymethyl)aminomethane, 0.067 M citric 241 acid monohydrate and 0.1 M boric acid; pH 6.0) and 1 ml of 0.5 M p-nitrophenyl-β-D- 242 glucopyranoside (CAS:2492-87-7) substrate; the mixture was incubated at 37 °C for 1 243 h, and the reaction was stopped by adding 1 ml of 0.5 M CaCl2 and 4 ml of 0.1 M 244 tris(hydroxymethyl)aminomethane (pH 12.0). Then, the mixture was filtered and 245 production of p-nitrophenol (PNP) was measured colourimetrically using a 246 spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan) at 410 nm. Activities of NAG 247 and acid/alkaline PME were measured as for BG activity, except we used a different 248 substrate and pH for the reaction system: we used p-nitrophenyl-N-acetyl-β-D- 249 glucosaminidine, p-nitrophenyl phosphate and p-nitrophenyl phosphate as substrates 250 buffered at pHs of 5.5 (Parham & Deng, 2000), 6.5 and 11.0 (Tabatabai et al., 1994), 251 respectively. Activities were expressed as the rate of PNP production (mg PNP kg soil- 1 252 h-1). We acknowledge that our enzyme “activity” measurements only provide an 253 indication of enzyme concentrations and do not represent actual soil enzyme activities 254 (Wallenstein and Weintraub 2008). 255 256 Statistical analyses 257 Data were tested for normality using the Kolmogorov-Smirnov test, and homogeneity 258 of variance was determined using Levene’s test; data were log-transformed for analysis 12 259 of variance (ANOVA) as appropriate. Three-way ANOVA, with N addition, rate of P 260 addition and chemical form of P fertilizer as factors, was used to test for treatment 261 differences in soil physicochemical properties and enzymatic activities. Associations 262 between enzymatic activities and physicochemical properties were tested using Pearson 263 correlation analysis. Statistical analyses were performed in SPSS 19.0 for Windows 264 (SPSS Inc., Chicago, USA). Redundancy analysis (RDA) (Canoco for Windows 5.0, 265 Plant Research International, Wageningen, The Netherlands) was used to determine the 266 relationships between soil physicochemical properties and enzymatic activities; prior 267 to analysis, enzymatic activity data were log10(x+1)-transformed to correct for positive 268 skewing, and the soil physicochemical properties were zero-centered for data 269 standardization. We used a Monte Carlo test (499 permutations) (Canoco for Windows 270 5.0, Plant Research International, Wageningen, The Netherlands) to determine the 271 relative contribution of soil parameters to variation in enzymatic activities, and the 272 relationship between BG, NAG and acid PME enzymatic activities was tested using 273 standardized major-axis (SMA) regression in R software (http://www.r-project.org, last 274 accessed: February 2018) and compared with regression slopes of unity to identify 275 isometric (not different from unity) or allometric (different from unity) relationships at 276 P < 0.05. 277 278 Results 279 Effects of N and P on soil physicochemical properties 280 Soil pH decreased with addition of Ca(H2PO4)2 under ambient and elevated levels of N 13 281 input (P < 0.01; Tables S1 and S2) due to release of H+ during hydrolysis of Ca(H2PO4)2. 282 There was a tendency towards a decrease in SOC concentration with increasing rate of 283 KH2PO4, irrespective of N treatment (P = 0.06). Addition of N increased TIN 284 concentration, regardless of form of P (P < 0.01; Table S2). TP concentration was 285 greater with increasing rate of KH2PO4, irrespective of addition of N, and with addition 286 of Ca(H2PO4)2, without N (Tables S1 and S2). Olsen-P concentration was greater with 287 increasing rate of P, irrespective of form of P and addition of N (P < 0.01; Table S2). 288 We found that MBP concentration was greater with increasing rate of added P, 289 regardless of form of P and addition of N (P < 0.01; Figures 1a and b, Table S1) and it 290 was greater under KH2PO4 than Ca(H2PO4)2 (P < 0.01; Table S3, Figure 1). In general, 291 there was a negative effect of N on AMF biomass (Figure S2a). 292 293 Effects of N and P on enzymatic activities 294 In general, addition of N positively affected BG, NAG and alkaline PME activities, 295 while there was an interaction between the effects of N and rate of P on acid PME 296 activity (Figure 2, Table S3). β-glucosidaseBG activity increased with higher rates of 297 KH2PO4 under ambient N (P < 0.01; Figure 2a) and at 4, 8 and 10 g m-2 y-1 KH2PO4 298 and 6 g m-2 y-1 Ca(H2PO4)2 under elevated N (Figures 2a and b). The higher rates of P 299 (6-10 g m-2 y-1) increased NAG activity, regardless of N and form of P (P < 0.01; Figures 300 2c and d). Acid PME activity was not affected by the addition of KH2PO4, irrespective 301 of N (Fig. 2e), and increased with addition of Ca(H2PO4)2 with elevated N (Figure 2f). 302 Alkaline PME activity increased with increasing rate of KH2PO4, irrespective of N 14 303 (Figure 2g) and with rate of Ca(H2PO4)2 under elevated N (P < 0.01; Figure 2h). 304 Chemical form of P affected alkaline PME activity (P < 0.05; Table S3), where average 305 activity across rates of P and N was greater with added Ca(H2PO4)2 than KH2PO4. We 306 found positive associations between BG and NAG (P < 0.01; Figure 3a, Table S4) and 307 between NAG and acid PME (P = 0.02; Figure 3f, Table S4), and all slopes in the SMA 308 regression differed from 1.0, indicating allometric relationships between enzymatic 309 activities (Table S4). 310 311 Effects of N and P on plant and soil function 312 Aboveground plant biomass increased with elevated N and, when averaged across rates 313 of P and N treatment, was greater with addition of KH2PO4 than Ca(H2PO4)2 (P = 0.036; 314 Figure S2b). Phosphorus uptake in the three plant species was greater with addition of 315 KH2PO4 than Ca(H2PO4)2 (P = 0.02; Figure S2c), with increasing rate of added P (P < 316 0.01) and with elevated N (P < 0.01). Addition of N and P resulted in greater levels of 317 aboveground litterfall biomass (Figures 4a and b). 318 β-glucosidaseBG activity was positively correlated with litterfall biomass, 319 regardless of form of P and addition of N (Figures 4c and d), while under addition of 320 KH2PO4, it was positively correlated with TP (Figure S3a) and Olsen P (Figure S3b) 321 concentrations and negatively associated with total C:P (P = 0.01; Figure S3c) and total 322 N:P (P = 0.01; Figure S3d) ratios. 323 N-acetyl-glucosaminidase NAG activity was positively correlated with TP (P < 324 0.01; Figure S3e) and Olsen P (P < 0.05; Figure S3f) and negatively correlated with 15 325 total C:P (P < 0.01; Figure S3g) and total N:P (P < 0.05; Figure S3h) ratios, under both 326 forms of P type, across N treatments. Acid PME activity was correlated negatively with 327 Olsen P concentration with addition of KH2PO4 and positively correlated in the 328 Ca(H2PO4)2 treatment (P < 0.05; Figure S3i). Alkaline PME activity was positively 329 correlated with Olsen P concentration with addition of KH2PO4 (P = 0.001; Figure S3j). 330 Under addition of KH2PO4, activities of BG, NAG and alkaline PME were 331 positively associated with the first axis of the RDA, together with plant P uptake, plant 332 biomass and TP, Olsen P and TIN concentrations; activity of acid PME was negatively 333 associated, together with SOC concentration (Figure 5a). Activities of BG, NAG and 334 acid PME under addition of Ca(H2PO4)2 were correlated with plant P uptake and Olsen 335 P and TP concentrations, whereas activity of alkaline PME was correlated with pH 336 (Figure 5b). Overall variation in enzymatic activities under the addition of KH2PO4 337 tended to be driven by plant P uptake, plant biomass and concentrations of Olsen-P, TP, 338 SOC and TIN that, together, explained 42.2% of the total variation (Fig. 5a). In contrast, 339 soil pH, plant P uptake and Olsen-P and TP concentrations explained 25.5% of the 340 variation in enzymatic activities under the addition of Ca(H2PO4)2 (Figure 5b). 341 342 Discussion 343 Microbial biomass PMBP concentration increased under the two chemical forms of P, 344 irrespective of addition of N, indicating immobilization of P in microbial biomass and 345 limitation of P in this typical meadow steppe ecosystem. It is likely that alleviation of 346 microbial P limitation would trigger the activity of extracellular enzymes, because our 16 347 multivariate analyses showed that P stocks and availability and plant P uptake were key 348 drivers of the increases in enzymatic activities. 349 350 Effects of P on enzymatic activities 351 Our study is one of few that have reported increases in BG activity in response to greater 352 availability of P, and thus far the only study in the cold to mid-temperate transitional 353 climatic zone (Table 1). We detected a positive effect of P on BG activity, indicating 354 soil-C cycling in this meadow steppe may be constrained by P availability; this finding 355 supported our first hypothesis. However, the effect of P depended on its chemical form, 356 because BG activity was greater with increasing rate of KH2PO4, but unaffected by 357 Ca(H2PO4)2. This finding was supported by the positive correlations of BG activity with 358 soil TP and with Olsen P concentrations when KH2PO4 was added, and the lack of such 359 associations under addition of Ca(H2PO4)2 (Figure S3a and b). It is also possible that 360 optimal BG activity decreased with the lower pH levels recorded under the addition of 361 Ca(H2PO4)2. The addition of the less soluble Ca(H2PO4)2 may have reduced 362 decomposition rates, because microbial BG activity was lower than in soils treated with 363 the more soluble KH2PO4. 364 Previous studies in wetland and alpine meadow soils found that BG activity was 365 unaffected by P loading availability, but positively correlated with DOC concentration 366 (Wright & Reddy, 2001; Jing et al., 2016). In our study, litterfall biomass, but not DOC 367 concentration, positively affected BG activity, regardless of form of P, indicating that 368 plant litter played a more important role than DOC concentration in the regulation of 17 369 BG activity. Higher Increased levels of soil N and P may increase improve substrate 370 quality, such as rlower educed litterfall C:N and C:P, and increase quantity (as litterfall 371 biomass) (Hobbie, 2005; Li et al., 2017). The negative correlations between soil C:P 372 and N:P with BG activity (Figures S3c and d) support the premise that substrate quality 373 plays an important role in the regulation of enzymatic activities (Wallenstein et al., 2009; 374 Phillips et al., 2014). Indeed, litter contains abundant cellulose and hemicellulose that 375 then serve as substrates and induce BG activity (Allison et al., 2013; Sinsabaugh et al., 376 2008); however, dissolved organic matter (including DOC) is a enzymatic product of 377 litter decomposition that may inhibit BG activity (Tian et al., 2010). We found that BG 378 activity was stimulated by the increased N and P inputs, likely due to the direct positive 379 roles of P and N availability in the synthesis of proteins and soil enzymes (Sinsabaugh 380 et al., 2014; Tian et al., 2016). 381 Addition of P led to an increase in microbial N demand, as indicated by the greater 382 levels of NAG activity (regardless of form of P), which support our first hypothesis. 383 Microbial NAG activity may eventually be subjected to soil C limitation in this meadow 384 steppe, because we found that increased application of the more soluble KH2PO4 385 decreased the concentration of SOC that was possibly linked to an increase in 386 decomposition. Indeed, addition of P also increased loss of soil C by increasing SOC 387 mineralization in Swedish meta-replicated long-term field experiments (Poeplau et al., 388 2016a, b). 389 The positive effects of soil P (TP and Olsen P) levels on BG and NAG activities 390 contrasted with the lack of effects reported from a meta-analysis of 17 studies of tropical 18 391 ecosystems (Waring et al., 2014). Paradoxically, microbial enzyme activities may be 392 constrained by P in relatively fertile chernozems, but not in highly weathered and P- 393 limited tropical soils, and it is possible these contrasting results may be due to 394 differences in data synthesis from large-scale ecosystems and small-scale field- 395 manipulative experiments (Niu et al., 2016). Contrasting correlations between Olsen P 396 concentration and acid PME activity under addition of KH2PO4 (negative) and 397 Ca(H2PO4)2 (positive) may have been due to the greater levels of plant P demand under 398 Ca(H2PO4)2 addition (Figure S2c) that are usually associated with low levels of Olsen 399 P and high levels of PME activities (Antibus et al., 1992). Soil P parameters have been 400 reported to positively (Colvan et al., 2001; Tian et al., 2016) and negatively (Olander 401 & Vitousek, 2000; Phoenix et al., 2004) affect PME activity, where responses may 402 depend on effects of initial levels of soil P, plant productivity, intensity of P uptake by 403 plants, and soil properties on abiotic P fixation (Tian et al., 2016; Margalef et al., 2017). 404 In our study, the increase in alkaline PME activity, even with exogenous P inputs, 405 indicated that microbial P demand was stimulated with nutrient addition. We found a 406 lack of response in aboveground biomass to addition of P (Figure S1b). Nevertheless, 407 the increase in microbial P demand and uptake, as supported by the observed rise in 408 MBP under fertilization with the two forms of P, could have diminished the ability of 409 root biomass to successfully outcompete microbes for P (Marschner et al., 2011). 410 Inconsistent changes in MBC concentrations and enzymatic activities indicate a 411 decoupling of the size and activity of the microbial community under elevated nutrient 412 inputs (Lori et al., 2017), and asymmetric changes in MBC with MBP concentrations 19 413 indicate that soil microorganisms may preferentially immobilize P (Bünemann et al., 414 2012) and are stoichiometrically plastic (Xu et al., 2013) in response to nutrient inputs. 415 Chemical form of P only affected alkaline PME activity, partially supporting our 416 second hypothesis, where we found greater levels of alkaline PME activity in the 417 Ca(H2PO4)2 treatment that were associated with lower levels of Olsen P. The RDA 418 indicated that drivers of enzymatic activity differed between the two forms of P (Figure 419 5) and the overall contrasting effects of P form were likely caused by differences in soil 420 environments and soil-plant interactions, such as the rate and intensity of P uptake. 421 422 Effects of N on enzymatic activities 423 We found that BG activity increased with elevated N, indicating that greater availability 424 of N alleviated microbial N limitation and stimulated microbial BG activity, thus 425 supporting our first hypothesis. Although evidence that availability of N increases BG 426 activity has been reported from other grassland and forest ecosystems (Henry et al., 427 2005; Keeler et al., 2009), our results contrast with those from semi-arid steppe 428 grasslands in Inner Mongolia, where BG activity decreased with N addition (Wei et al., 429 2013; Yang et al., 2017). This discrepancy may be due to differences in effects of 430 temperature, precipitation and soil fertility on the decomposition of plant residues and 431 supply of C to microorganisms. For example, the meadow steppe is less water-limited 432 than the semi-arid steppe, and an increase in soil moisture in Inner Mongolian 433 grasslands has been reported to alleviate soil acidification, due to a reduction in leaching 434 of basic cations (Cai et al., 2017), and physiological stress in soil microorganisms 20 435 caused by atmospheric N deposition (Zhang et al., 2015; Yang et al., 2017). Thus, 436 improved water conditions in wetter meadow steppes may interact with higher N 437 availability to promote microbial growth and BG activity. Our finding that acid PME 438 activity increased with elevated N indicated associated increases in P limitation and 439 microbial and plant demand for P. Given that mineralization of C is the first step in P 440 mineralization, where the hydrolysis of large C polymers facilitates the enzymatic 441 catalysis of P-C and N-C hydrolysis, it is likely that increased BG activity may lead to 442 subsequent P mineralization. 443 Although the increase in NAG activity with N addition was unexpected, positive 444 effects of N on NAG activity have been detected in bulk soil (Yang et al., 2017) and 445 soil fractions (Wang et al., 2015) in a semi-arid steppe ecosystem. The addition of N 446 may have enhanced plant N uptake that increased plant productivity (Hodge et al., 2000) 447 and microbial N demand. According with this, increases in the rates of litter 448 decomposition associated to soil enzyme activities (including N-cycle enzymes) has 449 been observed in response to N-addition (Wang et al., 2011). A recent meta data analysis 450 indicated how N fertilization increases the activities of hydrolase and oxidase enzymes, 451 related to an increase in litter production due to higher plant production under higher 452 levels of N-availability (Jian et al., 2016). Increased NAG activity may derive from 453 increases in mycorrhizal biomass for higher P transportation, possibly in response to 454 higher plant P demand under elevated N (Miller et al., 1998; Henry et al., 2005). 455 However, the increase in aboveground plant biomass (Figure S1b) coupled with a 456 decrease in AMF biomass (Figure S2a) under the addition of N indicated more effective 21 457 competition by plants for N, resulting in N-limitation among the soil microorganisms, 458 especially AMF, that then led to increased NAG production with greater plant density 459 and productivity. 460 The divergent responses of acid and alkaline PME activities to N addition in this 461 study may be due to greater levels of plant productivity (Figure S2b) and plant P uptake 462 (Figure S2c) and indicate that PME production may have derived from different sources; 463 for example, acid PME is produced by plant roots and soil microbes, whereas alkaline 464 PME is principally produced by soil microbes (Tabatabai, 1994). Therefore, stable acid 465 PME activity may be the consequence of a tradeoff between plant and soil microbial 466 demand for P due to N enrichment. The greater levels of alkaline PME activity under 467 N addition infer greater microbial P demand as a result of superior competition by plants 468 for P (Marschner et al., 2011), as supported by the greater levels of plant biomass and 469 plant P uptake (Figures S2b and c) and unaffected MBC (Table S2) and MBP 470 concentrations (Figure 1) in response to elevated N. 471 472 Stoichiometric traits of soil enzymes 473 The extracellular enzyme model (Moorhead et al., 2012) and data collected from 474 globally distributed soils and freshwater sediments (Sinsabaugh et al., 2009) have 475 demonstrated that the ratios of the activities of C-, N- and P-acquiring enzymes 476 approximately converge to 1. Usually, soil microbial growth is more limited by C than 477 N or P (Allison et al., 2010); however, enzymatic activity is not always correlated with 478 nutrient requirements for microbial growth, as indicated by our data. The SMA analysis 22 479 indicated that microbial activity in the grassland was more co-limited by N and P than 480 the global average (Figure 3; Table S4). Indeed, it has been shown that N limitation 481 constrains grassland productivity (Ren et al., 2017) and microbial activity (Henry et al., 482 2005), whereas P limitation of productivity may be gradual, as indicated by the globally 483 decreasing soil P pool across grassland soils, due to intensified forage production and 484 food supply (Sattari et al., 2012, 2016). Thus, N and P fertilization may be necessary to 485 maintain fertility in grassland soils (Sattari et al., 2012), because increases in 486 atmospheric N and P deposition may not be sufficient. Under this scenario, it is likely 487 that greater levels of large scalelarge-scale ecosystem nutrient inputs would facilitate 488 microbial activity that may then affect plant nutrition and soil C sequestration. The 489 optimal amounts of P addition in this grassland ecosystem is suggested to be 6 g P m-2 490 yr-1 as shown by the relatively higher extracellular enzyme activities and potentially 491 enhanced nutrient cycling rates at this P input level. Enzymatic stoichiometry may be a 492 more reliable indicator of microbial nutrient limitation than microbial biomass C:N:P 493 ratios (Xu et al., 2013), due to the functional role of enzymes in the uptake and cycling 494 of nutrients that sustain ecosystem productivity (Sinsabaugh & Shah, 2011). 495 496 Conclusions 497 Availability of N and P elicited positive effects on the activities of BG, NAG and 498 alkaline PME; alkaline PME activity was lower under the more soluble KH2PO4. 499 Elevated N input stimulated plant productivity and P uptake and led to soil microbial P 500 limitation that was greater in the Ca(H2PO4)2, as indicated by higher levels of alkaline 23 501 PME activity. Addition of N increased activities of BG, NAG and alkaline PME by 502 increasing substrate availability, potentially increasing plant-microbe competition for C 503 and N and intensity of plant P uptake. Our data indicated that KH2PO4 mediated changes 504 in enzymatic activities tended to be highly and positively associated with soil P 505 availability and intensity of plant P uptake, while Ca(H2PO4)2 mediated changes in soil 506 pH played a more essential role in enzymatic activities than plant P uptake. The 507 activities of soil enzymes in the study grassland were principally determined by P 508 availability and plant P content, indicating anthropogenic changes in ecosystem N and 509 P levels may elicit similar effects on soil enzymes, but that will likely depend on the 510 chemical form of P fertilizer. 511 512 Acknowledgements 513 This study was financially supported by the National Natural Science Foundation of 514 China (31770525), the National Key Research and Development Program of China 515 (2016YFC0500707, 2016YFC0500X01) and the European Research Council Synergy 516 grant (ERC-SyG-2013-610028, IMBALANCE-P). 517 518 Data accessibility 519 Data sets may be obtained from the corresponding author. 520 521 References 522 Allison, S.D., Weintraub, M.N., Gartner, T.B., Waldrop, M.P., 2010. Evolutionary- 24 523 economic principles as regulators of soil enzyme production and ecosystem 524 function. In: Shukla, G., Varma, A. (Eds). Soil enzymology. Springer-Verlag Berlin 525 Heidelberg, pp. 229-243. 526 Allison, S.D., Lu, Y., Weihe, C., Goulden, M.L., Martiny, A.C., Treseder, K.K., Martiny, 527 J.B., 2013. Microbial abundance and composition influence litter decomposition 528 response to environmental change. Ecology 94, 714-725. 529 Alvarez-Clare, S., Mack, M.C., Brooks, M., 2013. A direct test of nitrogen and 530 phosphorus limitation to net primary productivity in a lowland tropical wet 531 forest. Ecology 94, 1540–1551. 532 Bai, Y., Wu, J., Clark, C. M., Naeem, S., Pan, Q., Huang, J., Zhang, L., Han, X., 2010. 533 Tradeoffs and thresholds in the effects of nitrogen addition on biodiversity and 534 ecosystem functioning: evidence from inner Mongolia Grasslands. Global Change 535 Biology 16, 358-372. 536 Bilyera, N., Blagodatskaya, E., Yevdokimov, I., Kuzyakov, Y., 2018. Towards a 537 conversion factor for soil microbial phosphorus. European Journal of Soil Biology 538 87, 1-8. 539 Binkley, D., Högberg, P., 2016. Tamm review: revisiting the influence of nitrogen 540 deposition on Swedish forests. Forest Ecology and Management 368, 222-239. 541 Brookes, P.C., Powlson, D.S., Jenkinson, D.S., 1982. Measurement of microbial 542 biomass phosphorus in soil. Soil Biology & Biochemistry 14, 319-329. 543 Bünemann, E.K., Oberson, A., Liebisch, F., Keller, F., Annaheim, K.E., Huguenin-Elie, 544 O., Frossard, E., 2012. Rapid microbial phosphorus immobilization dominates 25 545 gross phosphorus fluxes in a grassland soil with low inorganic phosphorus 546 availability. Soil Biology & Biochemistry 51, 84-95. 547 Cai, J., Luo, W., Liu, H., Feng, X., Zhang, Y., Wang, R., Xu, Z., Zhang, Y., Jiang, Y., 548 2017. Precipitation-mediated responses of soil acid buffering capacity to long-term 549 nitrogen addition in a semi-arid grassland. Atmospheric Environment 170, 312- 550 318. 551 Carreiro, M.M., Sinsabaugh, R.L., Repert, D.A., Parkhurst, D.F., 2000. Microbial 552 enzyme shifts explain litter decay responses to simulated nitrogen 553 deposition. Ecology 81, 2359-2365. 554 Cenini, V.L., Fornara, D.A., McMullan, G., Ternan, N., Lajtha, K., Crawley, M.J., 2015. 555 Chronic nitrogen fertilization and carbon sequestration in grassland soils: evidence 556 of a microbial enzyme link. Biogeochemistry 126, 301-313. 557 Chen, J., Luo, Y., Li, J., Zhou, X., Cao, J., Wang, R. W., Wang, Y., Shelton, S., Jin, Z., 558 Walker L. M., Feng, Z., Niu, S., Feng, W., Jian, S., Zhou, L., 2017. Costimulation 559 of soil glycosidase activity and soil respiration by nitrogen addition. Global 560 Change Biology 23, 1328-133. 561 Colvan, S., Syers, J., O'Donnell, A., 2001. Effect of long-term fertiliser use on acid and 562 alkaline phosphomonoesterase and phosphodiesterase activities in managed 563 grassland. Biology and Fertility of Soils 34, 258-263. 564 Craine, J.M., Morrow, C., Stock, W.D., 2008. Nutrient concentration ratios and co- 565 limitation in South African grasslands. New Phytologist 179, 829-836. 566 Craine, J.M., Jackson, R.D., 2010. Plant nitrogen and phosphorus limitation in 98 North 26 567 American grassland soils. Plant and Soil 334, 73-84. 568 Fernández-Martínez, M., Vicca, S., Janssens, I.A., Sardans, J., Luyssaert, S., Campioli, 569 M., Chapin III, F.S., Ciais, P., Malhi, Y., Obersteiner, M., Papale, D., Piao, S., 570 Reichstein, M., Peñuelas, J., 2014. Nutrient availability as the key regulator of 571 global forest carbon balance. Nature Climate Change 4, 471-476. 572 Henry, H.A., Juarez, J.D., Field, C.B., Vitousek, P.M., 2005. Interactive effects of 573 elevated CO2, N deposition and climate change on extracellular enzyme activity 574 and soil density fractionation in a California annual grassland. Global Change 575 Biology 11, 1808-1815. 576 Hobbie, S.E., 2005. Contrasting effects of substrate and fertilizer nitrogen on the early 577 stages of litter decomposition. Ecosystems 8, 644-656. 578 Hodge, A., Robinson, D., Fitter, A., 2000. Are microorganisms more effective than 579 plants at competing for nitrogen? Trends in Plant Science 5, 304-308. 580 IUSS Working Group WRB: World Reference Base for Soil Resources 2014. 581 International soil classification system for naming soils and creating legends for 582 soil maps. World Soil Resources Reports No. 106, FAO, Rome, 2014. 583 Jian, S., Li, J., Chen, J., Wang, G., Mayes, M. A., Dzantor, K.E., Hui, D., Luo, Y., 2016. 584 Soil extracellular enzyme activities, soil carbon and nitrogen storage under 585 nitrogen fertilization: A meta-analysis. Soil Biology & Biochemistry 101, 32-43. 586 Jing, X., Yang, X., Ren, F., Zhou, H., Zhu, B., He, J.S., 2016. Neutral effect of nitrogen 587 addition and negative effect of phosphorus addition on topsoil extracellular 588 enzymatic activities in an alpine grassland ecosystem. Applied Soil Ecology 107, 27 589 205-213. 590 Keeler, B.L., Hobbie, S.E., Kellogg, L.E., 2009. Effects of long-term nitrogen addition 591 on microbial enzyme activity in eight forested and grassland sites: implications for 592 litter and soil organic matter decomposition. Ecosystems 12, 1-15. 593 Liebisch, F., Keller, F., Huguenin-Elie, O., Frossard, E., Oberson, A., Bünemann, E. K., 594 2014. Seasonal dynamics and turnover of microbial phosphorus in a permanent 595 grassland. Biology and Fertility of Soils 50, 465-475. 596 Li, H., Yang, S., Xu, Z., Yan, Q., Li, X., van Nostrand, J.D., He, Z., Yao, F., Han, X., 597 Zhou, J., Deng, Y., Jiang, Y., 2017. Responses of soil microbial functional genes to 598 global changes are indirectly influenced by aboveground plant biomass 599 variation. Soil Biology & Biochemistry 104, 18-29. 600 Lori, M., Symnaczik, S., Mäder, P., De Deyn, G., Gattinger, A., 2017. Organic farming 601 enhances soil microbial abundance and activity-A meta-analysis and meta- 602 regression. PLoS ONE 12, e0180442. 603 Margalef, O., Sardans, J., Fernández-Martínez, M., Molowny-Horas, R., Janssens, I.A., 604 Ciais, P., Goll, D., Richter, A., Obersteiner, M., Peñuelas, J., 2017. Global patterns 605 of phosphatase activity in natural soils. Scientific Reports 7, 1337. 606 Marschner, P., Crowley, D., Rengel, Z., 2011. Rhizosphere interactions between 607 microorganisms and plants govern iron and phosphorus acquisition along the root 608 axis–model and research methods. Soil Biology & Biochemistry 43, 883-894. 609 Mganga, K. Z., Razavi, B. S., Kuzyakov, Y., 2015. Microbial and enzymes response to 610 nutrient additions in soils of Mt. Kilimanjaro region depending on land 28 611 use. European Journal of Soil Biology 69, 33-40. 612 Marschner, P., Crowley, D., Rengel, Z., 2011. Rhizosphere interactions between 613 microorganisms and plants govern iron and phosphorus acquisition along the root 614 axis–model and research methods. Soil Biology & Biochemistry 43, 883-894. 615 Miller, M., Palojärvi, A., Rangger, A., Reeslev, M., Kjøller, A., 1998. The use of 616 fluorogenic substrates to measure fungal presence and activity in soil. Applied and 617 Environmental Microbiology 64, 613-617. 618 Moorhead, D.L., Lashermes, G., Sinsabaugh, R.L., 2012. A theoretical model of C-and 619 N-acquiring exoenzyme activities, which balances microbial demands during 620 decomposition. Soil Biology & Biochemistry 53, 133-141. 621 Mori, T., Lu, X., Aoyagi, R., Mo, J., 2018. Reconsidering the phosphorus limitation of 622 soil microbial activity in tropical forests. Functional Ecology 32:1145-1154. 623 Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination 624 of phosphate in natural waters. Analytica Chimica Acta 27, 31-36. 625 Nannipieri, P., Trasar-Cepeda, C., Dick, R.P., 2018. Soil enzyme activity: a brief history 626 and biochemistry as a basis for appropriate interpretations and meta-analysis. 627 Biology and Fertility of Soils 54, 11-19. 628 Niklas, K.J., 2006. A phyletic perspective on the allometry of plant biomass-partitioning 629 patterns and functionally equivalent organ-categories. New Phytologist 171, 27-40. 630 Niu, S., Classen, A. T., Dukes, J.S., Kardol, P., Liu, L., Luo, Y., Rustad, L., Sun, J., Tang, 631 J., Templer, P.H., Thomas, R.Q., Tian, D., Vicca, S., Wang, Y., Xia, J., Zaehle, S., 632 2016. Global patterns and substrate-based mechanisms of the terrestrial nitrogen 29 633 cycle. Ecology Letters 19, 697-709. 634 Olander, L.P., Vitousek, P.M., 2000. Regulation of soil phosphatase and chitinase 635 activity by N and P availability. Biogeochemistry 49, 175-190. 636 Parham, J.A., Deng, S.P., 2000. Detection, quantification and characterization of β- 637 glucosaminidase activity in soil. Soil Biology & Biochemistry 32, 1183-1190. 638 Peñuelas, J., Sardans, J., Rivas-ubach, A., Janssens, I.A., 2012. The human-induced 639 imbalance between C, N and P in Earth's life system. Global Change Biology 18, 640 3-6. 641 Peñuelas, J., Poulter, B.., Sardans, J., Ciais, P., van der Velde, M., Bopp, L., Boucher, 642 O., Godderis, Y., Hinsinger, P., Llusia, J., Nardin, E., Vicca, S., Obersteiner, M., 643 Janssens, I.A., 2013. Human-induced nitrogen-phosphorus imbalances alter natural 644 and managed ecosystems across the globe. Nature Communications 4, 2934. 645 Phillips, L.A., Ward, V., Jones, M.D., 2014. Ectomycorrhizal fungi contribute to soil 646 organic matter cycling in sub-boreal forests. The ISME Journal 8, 699. 647 Phoenix, G.K., Booth, R.E., Leake, J.R., Read, D.J., Grime, J.P., Lee, J.A., 2003. Effects 648 of enhanced nitrogen deposition and phosphorus limitation on nitrogen budgets of 649 semi‐natural grasslands. Global Change Biology 9, 1309-1321. 650 Poeplau, C., Bolinder, M.A., Kirchmann, H., Kätterer, T., 2016a. Phosphorus 651 fertilisation under nitrogen limitation can deplete soil carbon stocks: evidence from 652 Swedish meta-replicated long-term field experiments. Biogeosciences 13, 1119- 653 1127. 654 Poeplau, C., Herrmann, A.M., Kätterer, T., 2016b. Opposing effects of nitrogen and 30 655 phosphorus on soil microbial metabolism and the implications for soil carbon 656 storage. Soil Biology & Biochemistry 100, 83-91. 657 Ren, H., Xu, Z., Isbell, F., Huang, J., Han, X., Wan, S., Chen, S., Wang, R., Zeng, D., 658 Jiang, Y., Fang, Y., 2017. Exacerbated nitrogen limitation ends transient stimulation 659 of grassland productivity by increased precipitation. Ecological Monographs 87, 660 457-469. 661 Sardans, J., Peñuelas, J., Estiarte, M., 2007. Seasonal patterns of root-surface 662 phosphatase activities in a Mediterranean shrubland. Responses to experimental 663 warming and drought. Biology and Fertility of Soils 43, 779-786. 664 Sardans, J., Alonso, R., Janssens, I. A., Carnicer, J., Vereseglou, S., Rillig, M. C., 665 Fernández-Martínez, M., Sanders, T.G.M., Peñuelas, J., 2016. Foliar and soil 666 concentrations and stoichiometry of nitrogen and phosphorous across European 667 Pinus sylvestris forests: relationships with climate, N deposition and tree 668 growth. Functional Ecology 30, 676-689. 669 Schimel, J.P., Weintraub, M.N., 2003. The implications of exoenzyme activity on 670 microbial carbon and nitrogen limitation in soil: a theoretical model. Soil Biology 671 & Biochemistry 35, 549-563. 672 Shukla, G., Varma, A., 2011. Soil enzymology. Springer-Verlag, Berlin, Heidelberg. 673 Sinsabaugh, R. L., Lauber, C. L., Weintraub, M. N., Ahmed, B., Allison, S. D., 674 Crenshaw, C., Contosta, A.R., Cusack, D., Frey, S., Gallo, M.E., Gartner, T.B., 675 Hobbie, S.E., Holland, K., Keeler, B.L., Powers, J.S., Stursova, M., Takacs- 676 Vesbach, C., Waldrop, M.P., Wallenstein, M.D., Zak, D.R., Zeglin, L.H., 2008. 31 677 Stoichiometry of soil enzyme activity at global scale. Ecology Letters 11, 1252- 678 1264. 679 Sinsabaugh, R.L., Hill, B.H., Shah, J. J. F., 2009. Ecoenzymatic stoichiometry of 680 microbial organic nutrient acquisition in soil and sediment. Nature 462, 795-798. 681 Sinsabaugh, R.L., Shah, J.J.F., 2011. Ecoenzymatic stoichiometry of recalcitrant 682 organic matter decomposition: the growth rate hypothesis in 683 reverse. Biogeochemistry 102, 31-43. 684 Sinsabaugh, R.L., Belnap, J., Findlay, S.G., Shah, J.J.F., Hill, B.H., Kuehn, K.A., Kuske, 685 C.R., Litvak, M.E., Martinez, N.G., Moorhead, D.L., Warnock, D.D., 2014. 686 Extracellular enzyme kinetics scale with resource 687 availability. Biogeochemistry 121, 287-304. 688 Sattari, S.Z., Bouwman, A.F., Giller, K.E., van Ittersum, M.K., 2012. Residual soil 689 phosphorus as the missing piece in the global phosphorus crisis 690 puzzle. Proceedings of the National Academy of Sciences 109, 6348-6353. 691 Sattari, S.Z., Bouwman, A.F., Rodríguez, R.M., Beusen, A.H.W., Van Ittersum, M.K., 692 2016. Negative global phosphorus budgets challenge sustainable intensification of 693 grasslands. Nature Communications 7, 10696. 694 Sterkenburg, E., Clemmensen, K.E., Ekblad, A., Finlay, R.D., Lindahl, B.D., 2018. 695 Contrasting effects of ectomycorrhizal fungi on early and late stage decomposition 696 in a boreal forest. The ISME Journal 12, 2187-2197. 697 Tabatabai, M., 1994. Soil enzymes. In: Bottomley, P.S., Angle, J.S., Weaver, R.W. 698 (Eds.), Methods of soil analysis: Part 2-microbological and biochemical properties, 32 699 4th edn. Soil Science Society of America, Madison, pp 775-833. 700 Tian, L., Dell, E., Shi, W., 2010. Chemical composition of dissolved organic matter in 701 agroecosystems: correlations with soil enzyme activity and carbon and nitrogen 702 mineralization. Applied Soil Ecology 46, 426-435. 703 Tian, J., Wei, K., Condron, L. M., Chen, Z., Xu, Z., Chen, L., 2016. Impact of land use 704 and nutrient addition on phosphatase activities and their relationships with organic 705 phosphorus turnover in semi-arid grassland soils. Biology and Fertility of Soils 52, 706 675-683. 707 Trivedi, P., Delgado-Baquerizo, M., Trivedi, C., Hu, H., Anderson, I.C., Jeffries, T. C., 708 Zhou, J., Singh, B.K., 2016. Microbial regulation of the soil carbon cycle: evidence 709 from gene–enzyme relationships. The ISME Journal 10, 2593-2604. 710 Turner, B.L., Wright, S.J., 2014. The response of microbial biomass and hydrolytic 711 enzymes to a decade of nitrogen, phosphorus, and potassium addition in a lowland 712 tropical rain forest. Biogeochemistry 117, 115-130. 713 Vance, E.D., Brooks, P.C., Jenkinson, D.S., 1987. An extraction method for measuring 714 soil microbial biomass-C. Soil Biology & Biochemistry 19, 703-707. 715 Wallenstein, M.D., McMahon, S.K., Schimel, J.P., 2009. Seasonal variation in enzyme 716 activities and temperature sensitivities in Arctic tundra soils. Global Change 717 Biology 15, 1631-1639. 718 Wallenstein, M.D., Weintraub, M.N., 2008. Emerging tools for measuring and modeling 719 the in situ activity of soil extracellular enzymes. Soil Biology & Biochemistry 40, 720 2098-2106. 33 721 Wang, C., Han, G., Jia, Y., Feng, X., Guo, P., Tian, X., 2011. Response of litter 722 decomposition and related soil enzyme activities to different forms of nitrogen 723 fertilization in a subtropical forest. Ecological Research 26, 505-513. 724 Wang, D., Ba, L., 2008. Ecology of meadow steppe in northeast China. The Rangeland 725 Journal 30, 247-254. 726 Wang, R., Filley, T. R., Xu, Z., Wang, X., Li, M. H., Zhang, Y., Luo, W., Jiang, Y., 2014. 727 Coupled response of soil carbon and nitrogen pools and enzyme activities to 728 nitrogen and water addition in a semi-arid grassland of Inner Mongolia. Plant and 729 Soil 381, 323-336. 730 Wang, R., Dorodnikov, M., Yang, S., Zhang, Y., Filley, T. R., Turco, R. F., Zhang, Y., 731 Xu, Z., Li, H., Jiang, Y., 2015. Responses of enzymatic activities within soil 732 aggregates to 9-year nitrogen and water addition in a semi-arid grassland. Soil 733 Biology & Biochemistry 81, 159-167. 734 Wang, Y., Ciais, P., Goll, D., Huang, Y., Luo, Y., Wang, Y.P., Bloom, A.A., Broquet, G., 735 Hartmann, J., Peng, S., Peñuelas, J., Piao, S., Sardans, J., Stocker, B.D., Wang, R., 736 Zaehle, S., Zechmeister-Boltenstern, S., 2018. GOLUM-CNP v1.0: a data-driven 737 modeling of carbon, nitrogen and phosphorus cycles in major terrestrial 738 biomes. Geoscientific Model Development 11, 3903-3928. 739 Waring, B.G., Weintraub, S.R., Sinsabaugh, R.L., 2014. Ecoenzymatic stoichiometry 740 of microbial nutrient acquisition in tropical soils. Biogeochemistry 117, 101-113. 741 Wei, C., Yu, Q., Bai, E., Lü, X., Li, Q., Xia, J., Kardol, P., Liang, W., Wang, Z., Han, 742 X., 2013. Nitrogen deposition weakens plant–microbe interactions in grassland 34 743 ecosystems. Global Change Biology 19, 3688-3697. 744 Wright, A.L., Reddy, K.R., 2001. Phosphorus loading effects on extracellular enzyme 745 activity in Everglades wetland soils. Soil Science Society of America Journal 65, 746 588–595. 747 Xu, X., Thornton, P.E., Post, W.M., 2013. A global analysis of soil microbial biomass 748 carbon, nitrogen and phosphorus in terrestrial ecosystems. Global Ecology and 749 Biogeography 22, 737-749. 750 Xu, W., Luo, X.S., Pan, Y., Zhang, L., Tang, A. H., Shen, J.L., Zhang, Y., Li, K., Wu, 751 Q., Yang, D.W., Zhang, Y.Y., Xue, J., Li, W.Q., Li, Q.Q., Tang, L., Lu, S.H., Liang, 752 T., Tong, Y.A., Liu, P., Zhang, Q., Xiong, Z.Q., Shi, X.J., Wu, L.H., Shi, W.Q., Tian, 753 K., Zhong, X.H., Shi, K., Tang, Q.Y., Zhang, L.J., Huang, J.L., He, C.E., Kuang, 754 F.H., Zhu, B., Liu, H., Jin, X., Xin, Y.J., Shi, X.K., Du. E.Z., Dore, A.J., Tang, S., 755 Collett Jr., J.L., Goulding, K., Sun, Y.X., Ren, J., Zhang, F.S., Liu, X. (2015). 756 Quantifying atmospheric nitrogen deposition through a nationwide monitoring 757 network across China. Atmospheric Chemistry and Physics 15, 12345-12360. 758 Yang, S., Xu, Z., Wang, R., Zhang, Y., Yao, F., Zhang, Y., Turco, R.F., Jiang, Y., Zou, 759 H., Li, H., 2017. Variations in soil microbial community composition and 760 enzymatic activities in response to increased N deposition and precipitation in 761 Inner Mongolian grassland. Applied Soil Ecology 119, 275-285. 762 Zhang, N., Wan, S., Guo, J., Han, G., Gutknecht, J., Schmid, B., Yu, L., Liu, W., Bi, Y., 763 Wang, W., Ma, K., 2015. Precipitation modifies the effects of warming and nitrogen 764 addition on soil microbial communities in northern Chinese grasslands. Soil 35 765 Biology & Biochemistry 89, 12-23. 766 36 767 Table 1 Literature review of effects of phosphorus (P) addition on soil β-glucosidase 768 (BG), N-acetyl-glucosaminidase (NAG) and acid and alkaline phosphomonoesterases 769 (PMEs) activity in grassland ecosystems. Acid Alkaline Grassland type and location P form BG NAG Reference PME PME Triple Meadow grassland, UK ￚ ￚ 0 ￪ Colvan et al. 2001 superphosphate Bünemann et al. Mesic grassland, Switzerland Superphosphate ￚ ￚ ￬ ￚ 2012 Mesic grassland, Switzerland Superphosphate ￚ ￚ ￬ ￚ Liebisch et al. 2014 Savannah, Tanzania KH2PO4 ￪ ￪ ￪ ￚ Mganga et al. 2015 Semi-natural grassland, Tanzania KH2PO4 ￪ ￪ ￪ ￚ Triple Alpine grassland, China 0 0 0 ￚ Jing et al. 2016 superphosphate Semi-arid steppe, China Superphosphate ￚ ￚ ￪ ￪ Tian et al. 2016 Old field, China Superphosphate ￚ ￚ ￬ ￬ 770 Effects annotated as ↑, ↓, 0 or - indicate positive, negative, no significant change or lack of data, 771 respectively. 37 772 Figure captions 773 Figure 1 Effect of addition of KH2PO4 (a) or Ca(H2PO4)2 (b) with 0 and 10 g N m-2 y- 1 774 nitrogen (N) on concentration of microbial biomass phosphorus (MBP) (mean ±SE, n 775 = 5). Upper- and lowercase letters indicate differences among KH2PO4 and Ca(H2PO4)2 776 treatments with and without added N, respectively. 777 778 Figure 2 Boxplots of activity of BG (a, b), NAG (c, d), acid PME (e, f) and alkaline 779 PME (g, h) with addition of KH2PO4 (a, c, e, g) and Ca(H2PO4)2 (b, d, f, h) with 0 (blue) 780 and 10 g N m-2 y-1 (red). Different letters indicate differences among KH2PO4 and 781 Ca(H2PO4)2 treatments with or without added N, and asterisks indicate differences 782 between N treatments for the rates of KH2PO4 and Ca(H2PO4)2. Error bars indicate the 783 10th and 90th percentiles; black lines within the boxes represent median activity and 784 the box limits indicate activity within the 25-75th percentile range. 785 786 Figure 3 Regression analyses of activities of NAG and BG, PME and BG and PME 787 and NAG. All data are Ln-transformed. Dashed line: line of unity. 788 789 Figure 4 Mean litter biomass (±SE, n = 5) with addition of KH2PO4 (a) or Ca(H2PO4)2 790 (b) under ambient and added nitrogen (N). Relationship between BG activity and litter 791 biomass with addition of KH2PO4 (c) or Ca(H2PO4)2 across N treatments (d). Upper- 792 and lowercase letters indicate differences among KH2PO4 and Ca(H2PO4)2 treatments 793 with and without N addition, respectively. Asterisk indicates within P rate and type 38 794 differences between N treatments. 795 796 Figure 5 Redundancy analysis of the relationship between soil enzyme activity (BG, 797 NAG, acid PME and alkaline PME) and explanatory parameters (plant P uptake, pH, 798 plant biomass and TP, TIN, SOC and Olsen-P concentrations) (left) and their 799 contributions to the variation in overall activity (right) under addition of KH2PO4 (a) or 800 Ca(H2PO4)2 (b) addition. 801 39 802 803 Figure 1 804 40 805 806 Figure 2 807 41 808 809 Figure 3 810 42 811 812 Figure 4 813 43 814 815 Figure 5 44