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Soil enzyme activity can be affected by both production and degradation processes, as enzymes can be degraded by proteases. However, the impact of nutrient addition on enzyme activity is often solely attributed to changes in enzyme production without fully considering degradation. In this study, we demonstrate that the activities of β-1,4-glucosidase (BG), β-D-cellobiohydrolase (CBH), β-1,4-xylosidase (BX), and β-1,4-N-acetyl-glucosaminidase (NAG) in two tropical plantations exhibited comparable levels between nitrogen (N)- and phosphorus (P)-fertilized soils and the unfertilized control under field conditions. However, it was observed that the reduction in enzymatic activity was significantly higher in the fertilized soils during short-term laboratory incubation in the acacia plantation. Additionally, the eucalyptus plantation exhibited a similar tendency, although statistical significance was not achieved due to the high variance of the data. The results show that the interruption of the natural, continuous supply of organic matter or non-soil microbial-derived enzymes, which typically occurs under field conditions, leads to a more significant reduction in soil enzyme activities in fertilized soils compared to unfertilized control. This may be attributed to the higher abundance of protease in fertilized soils, resulting in faster enzyme degradation. Interestingly, P fertilization alone did not have a similar effect, indicating that N fertilization is likely the main cause of the larger decreases in enzyme activity during incubation in fertilized soils compared to unfertilized control soils, despite our study site being poor in P and rich in N. These findings highlight the importance of considering enzyme degradation when investigating material dynamics in forest ecosystems, including the impact of nutrient addition on enzyme activity, as enzyme production alone may not fully explain changes in soil enzyme activity.

The effects of deposited nitrogen (N) on forest ecosystems largely depend on the amount of N retained in the ecosystems and its partitioning among ecosystem pools. However, our understanding of the capacity of tropical plantations to retain deposited N is limited. To evaluate the retention of deposited N in a human-disturbed pine plantation in southern China and compare the result with previous findings in an adjacent old-growth forest, we added 15N-tracer monthly to the forest floor for one year and determined its recovery in ecosystem compartments four months after the last addition. We monitored 15N recoveries in soil solution monthly to quantify leaching losses. The pine forest retained about 58 ± 5% of the 15N-labeled deposited N, which is lower than that reported in the adjacent old-growth forest (72 ± 6%). Both forests experience chronic N deposition (recently measured at 51 kg N ha−1 yr−1) and we attribute the difference in retention to effects of previous disturbance mainly understory and litter harvesting in the pine plantation. Only 3 kg N ha−1 yr−1 (5% of the 15N-labeled deposited N) out of the measured total leaching (54 kg N ha−1 yr−1) originated from deposited (and labeled) N from the measurement year, suggesting that N leaching is dominated by unlabeled N sources. Furthermore, results from our study and other similar 15N labeling experiments together show similar patterns of total ecosystem retention of deposited N in tropical and temperate forests, but here we demonstrate a decreasing retention of N with increased N deposition in these forests. Our findings indicate that plantation forests that experience human-disturbance and chronic N deposition have lower N retention compared to old-growth forests, and thus elevated N inputs in such ecosystems can cause risk of hydrological N losses, soil acidification, and freshwater pollution.

Biological nitrogen (N) fixation plays an important role in terrestrial N cycling and represents a key driver of terrestrial net primary productivity (NPP). Despite the importance of N fixation in terrestrial ecosystems, our knowledge regarding the controls on terrestrial N fixation remains poor. Here, we conducted a meta-analysis (based on 852 observations from 158 studies) of N fixation across three types of ecosystems with different status of disturbance (no management, restoration [previously disturbed], and disturbance [currently disturbed]) and in response to multiple environmental change factors (warming, elevated carbon dioxide [CO2 ], increased precipitation, increased drought, increased N deposition, and their combinations). We explored the mechanisms underlying the changes in N fixation by examining the variations in soil physicochemical properties (bulk density, texture, moisture, and pH), plant and microbial characteristics (dominant plant species numbers, plant coverage, and soil microbial biomass), and soil resources (total carbon, total N, total phosphorus (P), inorganic N, and inorganic P). Human disturbance inhibited non-symbiotic N fixation but not symbiotic N fixation. Terrestrial N fixation was stimulated by warming (+152.7%), elevated CO2 (+19.6%), and increased precipitation (+73.1%) but inhibited by increased drought (-30.4%), N deposition (-31.0%), and combinations of available multiple environmental change factors (-14.5%), the extents of which varied among biomes and ecosystem compartments. Human disturbance reduced the N fixation responses to environmental change factors, which was associated with the changes in soil physicochemical properties (2%-56%, p < .001) and the declines in plant and microbial characteristics (3%-49%, p ≤ .003) and soil resources (6%-48%, p ≤ .03). Overall, our findings reveal for the first time the effects of multiple environmental change factors on terrestrial N fixation and indicate the role of human disturbance activities in inhibiting N fixation, which can improve our understanding, modeling, and prediction of terrestrial N budgets, NPP, and ecosystem feedbacks under global change scenarios.

A mechanistic understanding of plant photosynthetic response is needed to reliably predict changes in terrestrial carbon (C) gain under conditions of chronically elevated atmospheric nitrogen (N) deposition. Here, using 2,683 observations from 240 journal articles, we conducted a global meta-analysis to reveal effects of N addition on 14 photosynthesis-related traits and affecting moderators. We found that across 320 terrestrial plant species, leaf N was enhanced comparably on mass basis (Nmass , +18.4%) and area basis (Narea , +14.3%), with no changes in specific leaf area or leaf mass per area. Total leaf area (TLA) was increased significantly, as indicated by the increases in total leaf biomass (+46.5%), leaf area per plant (+29.7%), and leaf area index (LAI, +24.4%). To a lesser extent than for TLA, N addition significantly enhanced leaf photosynthetic rate per area (Aarea , +12.6%), stomatal conductance (gs , +7.5%), and transpiration rate (E, +10.5%). The responses of Aarea were positively related with that of gs , with no changes in instantaneous water-use efficiency and only slight increases in long-term water-use efficiency (+2.5%) inferred from 13 C composition. The responses of traits depended on biological, experimental, and environmental moderators. As experimental duration and N load increased, the responses of LAI and Aarea diminished while that of E increased significantly. The observed patterns of increases in both TLA and E indicate that N deposition will increase the amount of water used by plants. Taken together, N deposition will enhance gross photosynthetic C gain of the terrestrial plants while increasing their water loss to the atmosphere, but the effects on C gain might diminish over time and that on plant water use would be amplified if N deposition persists.

In-situ soil cadmium (Cd) immobilization helps to reduce Cd accumulation in rice grain, while its effects on bioaccessibility of Cd in rice during digestion and the associated health risk from rice consumption remain unclear. Here, we combined in-situ soil Cd immobilization and bioaccessibility-corrected health risk assessment (HRA) to minimize both the risk and uncertainty of Cd exposure from rice consumption. Wollastonite with or without four different phosphates (P) were applied to immobilize soil Cd at paddy fields, and their influences on Cd, essential elements, and amino acids in rice grain were analyzed. Moreover, a bioaccessibility-corrected HRA was conducted to accurately reflect the Cd exposure risk from ingesting these rices. The results showed the co-application of wollastonite and four different P reduced Cd concentrations in rice grain equally, while their impacts on bioaccessibility of Cd in rice during simulated human digestion were inconsistent (53-71%). The HRA based on bioaccessibility of Cd in rice revealed that Cd exposure risk from rice consumption was lowest with the application of wollastonite, followed by the co-application of wollastonite and sodium hexametaphosphate. This work highlights the value of bioaccessibility-corrected HRA for screening the optimal Cd immobilization strategy to achieve safer rice consumption.

A massive rise in atmospheric nitrogen deposition (ND) has threatened ecosystem health through accelerating soil nitrogen (N) cycling rates. While soil microbes serve a crucial function in soil N transformation, it remains poorly understood on how excess ND affects microbial functional populations regulating soil N transformation in tropical forests. To address this gap, we conducted 13-year N (as NH

Soil fungi play critical roles in ecosystem processes and are sensitive to global changes. Elevated atmospheric nitrogen (N) deposition has been well documented to impact on fungal diversity and community composition, but how the fungal community assembly responds to the duration effects of experimental N addition remains poorly understood. Here, we aimed to investigate the soil fungal community variations and assembly processes under short- (2 years) versus long-term (13 years) exogenous N addition (∼100 kg N ha–1 yr–1) in a N-rich tropical forest of China. We observed that short-term N addition significantly increased fungal taxonomic and phylogenetic α-diversity and shifted fungal community composition with significant increases in the relative abundance of Ascomycota and decreases in that of Basidiomycota. Short-term N addition also significantly increased the relative abundance of saprotrophic fungi and decreased that of ectomycorrhizal fungi. However, unremarkable effects on these indices were found under long-term N addition. The variations of fungal α-diversity, community composition, and the relative abundance of major phyla, genera, and functional guilds were mainly correlated with soil pH and NO3––N concentration, and these correlations were much stronger under short-term than long-term N addition. The results of null, neutral community models and the normalized stochasticity ratio (NST) index consistently revealed that stochastic processes played predominant roles in the assembly of soil fungal community in the tropical forest, and the relative contribution of stochastic processes was significantly increased by short-term N addition. These findings highlighted that the responses of fungal community to N addition were duration-dependent, i.e., fungal community structure and assembly would be sensitive to short-term N addition but become adaptive to long-term N enrichment.

Anthropogenic elevated nitrogen (N) deposition has an accelerated terrestrial N cycle, shaping soil carbon dynamics and storage through altering soil organic carbon mineralization processes. However, it remains unclear how long-term high N deposition affects soil carbon mineralization in tropical forests. To address this question, we established a long-term N deposition experiment in an N-rich lowland tropical forest of Southern China with N additions such as NH4NO3 of 0 (Control), 50 (Low-N), 100 (Medium-N) and 150 (High-N) kg N ha−1 yr−1, and laboratory incubation experiment, used to explore the response of soil carbon mineralization to the N additions therein. The results showed that 15 years of N additions significantly decreased soil carbon mineralization rates. During the incubation period from the 14th day to 56th day, the average decreases in soil CO2 emission rates were 18%, 33% and 47% in the low-N, medium-N and high-N treatments, respectively, compared with the Control. These negative effects were primarily aroused by the reduced soil microbial biomass and modified microbial functions (e.g., a decrease in bacteria relative abundance), which could be attributed to N-addition-induced soil acidification and potential phosphorus limitation in this forest. We further found that N additions greatly increased soil-dissolved organic carbon (DOC), and there were significantly negative relationships between microbial biomass and soil DOC, indicating that microbial consumption on soil-soluble carbon pool may decrease. These results suggests that long-term N deposition can increase soil carbon stability and benefit carbon sequestration through decreased carbon mineralization in N-rich tropical forests. This study can help us understand how microbes control soil carbon cycling and carbon sink in the tropics under both elevated N deposition and carbon dioxide in the future.

Human activities have disturbed global phosphorus (P) cycling by introducing substantial amounts of P to natural ecosystems. Although natural P gradients and fertilization studies have found that plant community traits are closely related to P availability, it remains unclear how increased P supply affects plant growth and diversity in P-deficient tropical forests. We used a decadal P-addition experiment (2007–2017) to study the effects of increased P input on plant growth and diversity in understory layer in tropical forests. We monitored the dynamics of seedling growth, survival rate, and diversity of understory plants throughout the fertilization period under control and P addition at 15 g P m−2 yr−1. To identify the drivers of responses, P concentration, photosynthesis rate and nonstructural carbon were analyzed. Results showed that long-term P addition significantly increased P concentrations both in soil pools and plant tissues. However, P addition did not increase the light-saturated photosynthesis rate or growth rate of the understory plants. Furthermore, P addition significantly decreased the survival rate of seedlings and reduced the species richness and density of understory plants. The negative effects of P addition may be attributed to an increased carbon cost due to the tissue maintenance of plants with higher P concentrations. These findings indicate that increased P supply alone is not necessary to benefit the growth of plants in ecosystems with low P availability, and P inputs can inhibit understory plants and may alter community composition. Therefore, we appeal to a need for caution when inputting P to tropical forests ecosystems.

Significance Forest soil carbon (C) storage plays a central role in sequestrating atmospheric CO2 on timescales from centuries to millennia. However, our current understanding of soil C sequestration in response to N deposition mainly focuses on mid-to-high latitudes in the Northern Hemisphere, where N supply typically constrains forest growth. We lack data about changes in soil C stocks in tropical forests, where most ecosystems are N-rich or N-saturated. Using more than a decade of continuous N addition experiment and a meta-analysis, we found that excess N deposition can significantly increase soil C in N-rich tropical forests. However, enhanced C sequestration in tropical soils is not a good reason to justify excess N emissions to the atmosphere., Terrestrial ecosystem carbon (C) sequestration plays an important role in ameliorating global climate change. While tropical forests exert a disproportionately large influence on global C cycling, there remains an open question on changes in below-ground soil C stocks with global increases in nitrogen (N) deposition, because N supply often does not constrain the growth of tropical forests. We quantified soil C sequestration through more than a decade of continuous N addition experiment in an N-rich primary tropical forest. Results showed that long-term N additions increased soil C stocks by 7 to 21%, mainly arising from decreased C output fluxes and physical protection mechanisms without changes in the chemical composition of organic matter. A meta-analysis further verified that soil C sequestration induced by excess N inputs is a general phenomenon in tropical forests. Notably, soil N sequestration can keep pace with soil C, based on consistent C/N ratios under N additions. These findings provide empirical evidence that below-ground C sequestration can be stimulated in mature tropical forests under excess N deposition, which has important implications for predicting future terrestrial sinks for both elevated anthropogenic CO2 and N deposition. We further developed a conceptual model hypothesis depicting how soil C sequestration happens under chronic N deposition in N-limited and N-rich ecosystems, suggesting a direction to incorporate N deposition and N cycling into terrestrial C cycle models to improve the predictability on C sink strength as enhanced N deposition spreads from temperate into tropical systems.

Phosphorus (P) addition could largely alter soil microbial activity. However, effects of long-term P addition on soil extracellular enzyme activity are not well understood in tropical forests. To address this question, we measured absolute activities (activity per unit of dry soil) and specific activities (activity per unit of microbial biomass carbon) of enzymes involved in carbon (C), nitrogen (N), and P cycling in a 10-year P addition experimental site in a tropical forest. Phosphorus addition decreased acid phosphatase absolute and specific activity by 37% and 47%, respectively. As to N-acquisition enzymes, P addition increased leucine amino peptidase absolute activity but decreased β-1,4-N-acetylglucosaminidase absolute activity by 33%. Meanwhile, P addition had no effects on leucine amino peptidase specific activity but decreased β-1,4-N-acetylglucosaminidase specific activity by 43%. Among C-acquisition enzymes, cellobiohydrolase, α-glucosidase, and β-glycosidase absolute and specific activities showed no significant responses to P addition, while P addition decreased β-xylosidase absolute and specific activity by 15% and 27% respectively. Phosphorus addition also decreased phenol oxidase absolute activity by 30% and peroxidase absolute activity by 29%. These results suggest a strong P shortage for microorganisms and that P addition could decline microbial productions of enzymes in phosphorus-poor tropical forests. Results from this study emphasize again the important role of available P in tropical forests.

Nitrogen (N) deposition is a component of global change that has considerable impact on belowground carbon (C) dynamics. Plant growth stimulation and alterations of fungal community composition and functions are the main mechanisms driving soil C gains following N deposition in N-limited temperate forests. In N-rich tropical forests, however, N deposition generally has minor effects on plant growth; consequently, C storage in soil may strongly depend on the microbial processes that drive litter and soil organic matter decomposition. Here, we investigated how microbial functions in old-growth tropical forest soil responded to 13 years of N addition at four rates: 0 (Control), 50 (Low-N), 100 (Medium-N), and 150 (High-N) kg N ha-1 year-1 . Soil organic carbon (SOC) content increased under High-N, corresponding to a 33% decrease in CO2 efflux, and reductions in relative abundances of bacteria as well as genes responsible for cellulose and chitin degradation. A 113% increase in N2 O emission was positively correlated with soil acidification and an increase in the relative abundances of denitrification genes (narG and norB). Soil acidification induced by N addition decreased available P concentrations, and was associated with reductions in the relative abundance of phytase. The decreased relative abundance of bacteria and key functional gene groups for C degradation were related to slower SOC decomposition, indicating the key mechanisms driving SOC accumulation in the tropical forest soil subjected to High-N addition. However, changes in microbial functional groups associated with N and P cycling led to coincidentally large increases in N2 O emissions, and exacerbated soil P deficiency. These two factors partially offset the perceived beneficial effects of N addition on SOC storage in tropical forest soils. These findings suggest a potential to incorporate microbial community and functions into Earth system models considering their effects on greenhouse gas emission, biogeochemical processes, and biodiversity of tropical ecosystems.

Soil ecoenzymes play an important role in the decomposition and formation of soil organic matter, and are often considered to be derived from soil microbial and plant root activities. This study found another potentially-important source, transfer of ecoenzymes in phyllosphere to soil via throughfall. Throughfall samples were collected in five subtropical forests, and six ecoenzymes were determined therein. Data from this study showed that the ecoenzyme activity in throughfall was significantly higher than that in rainfall. Although this study has the limitation of a single measurement, it is the first report of the potential importance of ecoenzymes in throughfall in forest ecosystems.

Biological nitrogen (N) fixation (BNF), an important source of N in terrestrial ecosystems, plays a critical role in terrestrial nutrient cycling and net primary productivity. Currently, large uncertainty exists regarding how nutrient availability regulates terrestrial BNF and the drivers responsible for this process. We conducted a global meta-analysis of terrestrial BNF in response to N, phosphorus (P), and micronutrient (Micro) addition across different biomes (i.e, tropical/subtropical forest, savanna, temperate forest, grassland, boreal forest, and tundra) and explored whether the BNF responses were affected by fertilization regimes (nutrient-addition rates, duration, and total load) and environmental factors (mean annual temperature [MAT], mean annual precipitation [MAP], and N deposition). The results showed that N addition inhibited terrestrial BNF (by 19.0% (95% confidence interval [CI]: 17.7%-20.3%); hereafter), Micro addition stimulated terrestrial BNF (30.4% [25.7%-35.3%]), and P addition had an inconsistent effect on terrestrial BNF, i.e., inhibiting free-living N fixation (7.5% [4.4%-10.6%]) and stimulating symbiotic N fixation (85.5% [25.8%-158.7%]). Furthermore, the response ratios (i.e., effect sizes) of BNF to nutrient addition were smaller in low-latitude (30°) biomes (8.5%-36.9%) than in mid-/high-latitude (≥30°) biomes (32.9%-61.3%), and the sensitivity (defined as the absolute value of response ratios) of BNF to nutrients in mid-/high-latitude biomes decreased with decreasing latitude (p ≤ 0.009; linear/logarithmic regression models). Fertilization regimes did not affect this phenomenon (p 0.05), but environmental factors did affect it (p 0.001) because MAT, MAP, and N deposition accounted for 5%-14%, 10%-32%, and 7%-18% of the variance in the BNF response ratios in cold (MAT 15°C), low-rainfall (MAP 2,500 mm), and low-N-deposition (7 kg ha

In tropical plantations, nutrients such as nitrogen (N) or phosphorus (P) are often applied as management practices. However, the effects of such nutrient additions on topsoil C- and N-acquiring enzymes activities are unclear. In this study, the impacts of fertilization on β-1,4-glucosidase (BG), β-D-cellobiosidase (CBH), β-1,4-xylosidase (BX), β-1,4-N-acetyl-glucosaminidase (NAG), and leucine amino peptidase (LAP) enzymes activities from topsoil and litter layer of two tropical plantations (Acacia auriculiformis and Eucalyptus urophylla) were measured. The results showed that N addition had neutral impact on topsoil enzymes, while significantly elevating the activities of BG, CBH, BX, and NAG in the litter layer. P fertilization had no impacts except for an elevation of NAG in litter sample. There was no interactions found between N and P additions on these enzyme activities. The clearer impacts of N over P fertilization were unexpected because that the study site receives a high rate of atmospheric N deposition, and has low soil P availability. The impact of P fertilization on hydrolytic enzyme activities may be less important compared with that of N.

Asymbiotic nitrogen (N) fixation (ANF) is an important source of N in pristine forests and is predicted to decrease with N deposition. Previous studies revealing N fixation in response to N deposition have mostly applied understory N addition approaches, neglecting the key processes (for example, N retention and uptake) occurring in forest canopy. This study evaluated the effects of N deposition on N fixation in the soil, forest floor, mosses, and canopy leaves in a temperate forest (in central China) and a tropical forest (in southern China) with different treatments: control, understory N addition, and canopy N addition. Results showed that total ANF rates were higher in the temperate forest (2.57 ± 0.19 mg N m−2 d−1) than in the tropical forest (1.34 ± 0.09 mg N m−2 d−1). N addition inhibited the soil, forest floor, moss, and foliar N fixation in the temperate forest, whereas it inhibited only the soil N fixation in the tropical forest. Compared to canopy N addition, understory N addition overestimated the inhibitory effects of N deposition on total ANF slightly in the tropical forest (by 35%) but severely in the temperate forest (by 375–472%) due to neglecting canopy retention of N. In summary, our findings indicate that ANF has different rates and sensitivities to N addition between tropical and temperate forests and that understory N addition overestimates the N deposition effects on ANF in forests, particularly in the temperate forest. These findings are important for our accurate understanding and estimate of terrestrial N fixation under N deposition scenarios.

Increased nitrogen (N) deposition endangers the biodiversity and stability of forest ecosystems, and much of the original phosphorus (P) parent material continues to decrease in most lowland tropical forests. It remains poorly understood as to how soil microbial diversity at a molecular level responds to the addition of excess N and mitigation of soil P limitation, as well as their influencing factors, in the N-rich tropical forest ecosystems. To reach a better understanding, we conducted a six-year N and P-addition experiment consisting of three treatments: N-addition (150 kg N ha−1 yr−1), P-addition (150 kg P ha−1 yr−1), and NP-addition (150 kg N ha−1 yr−1 plus 150 kg P ha−1 yr−1), besides a control treatment in an old-growth tropical forest in southern China. We examined the snapshot responses of soil bacterial richness and community composition to the elevated N and P levels after six years using a 16S rRNA gene MiSeq sequencing method. The soil bacterial α-diversity, which is represented by Chao1 index in terms of bacterial richness, was 783 ± 87 (mean ± SD) across all samples in this study. The N addition caused a decline in soil bacterial richness, most likely through its negative effect on soil pH. The decrease in soil pH resulted from the direct N input and indirect NO3− increase. However, the P treatment had no effect on soil bacterial richness. The NP treatment also reduced the soil bacterial richness as the N addition. These results suggested that the P input could not alleviate the loss of soil bacterial richness induced by excess N deposition in the old-growth N-rich tropical forest. The Acidobacteria, which comprised 31.1% of the soil bacterial community, were the most dominant bacteria across all samples. The addition of P shifted the soil bacterial community composition. The elevated P availability with P-addition and the decreased understory plant coverage in the N-input treatment altered the soil bacterial β-diversity. Our results highlight the different roles of N and P depositions in shaping the soil bacterial richness and community composition, thereby causing concomitant changes in understory plant and underground microbial communities in this ecosystem.

Because potassium (K) is a rock-derived essential element that can be depleted in highly-weathered tropical soils, K availability may limit some portion of soil microbial activity in tropical forest ecosystems. In this paper we tested if K limits microbial activity in the condition of sufficient labile C supply. An incubation experiment was performed using surface soil samples (0–10 cm depth) obtained from four permanent ecological research plots in a natural sub-tropical forest in southern China. Soil samples were taken in September 2016. Heterotrophic soil respiration rates and microbial biomass were measured after the addition of glucose (both D and L) with and without K (potassium chloride). We did not observe any effects of K addition on soil microbial respiration, suggesting that K does not limit the microbial activity in the condition of sufficient labile C supply. The lack of microbial response to added K can be attributed to the high mobility of K in forest ecosystems, which may have provided sufficient K to microbes in our soil samples (already provided at the beginning of the incubation). However, at the present stage, we cannot conclude that K is not a limiting factor of soil microbial activity in other tropical forest ecosystems because of the heterogeneity of tropical forest ecosystems and few observations. The hypothesis needs to be tested in larger numbers of tropical forests.

The ratio of β-1,4-glucosidase (BG) to β-1,4-N-acetylglucosaminidase (NAG) activity (BG:NAG ratio) is often used as an indicator of the relative resource allocation of soil microbes to C acquisition compared with N. An increasing number of recent studies have used this index to assess the nutrient status of microbes. However, the validity of this index for assessing the nutrient status of microbes is not well tested. In this study, we collected published data and tested that validity by investigating whether N fertilization elevated the BG:NAG ratio, assuming that microbes reduce their allocation to the N-acquiring enzyme (NAG) under N-enriched conditions. Of the data points, 54% (82/151) did not support the hypothesis because those studies showed lower BG:NAG ratios in N-enriched soils than under ambient conditions, especially when the ambient BG:NAG ratio was higher than 2.0 (77%, 59/77). This suggests that the BG:NAG ratio does not always indicate the microbial status for C or N limitation. Rather, we hypothesized that the decomposition stage explained the variation in BG:NAG because N addition accelerates decomposition, and the BG:NAG ratio is lower at later stages of decomposition due to the dominance of NAG-targeting C (chitin or peptidoglycan). A negative correlation of BG:NAG ratio with polyphenol oxidase activity, which increases with decomposition, supported our hypothesis.

Background: Soil fungi play critical roles in ecosystem processes and are sensitive to global changes. Elevated atmospheric nitrogen (N) deposition has been well documented to impact on fungal diversity and community composition, but how fungal community assembly respond to short- and long-term simulative N deposition remains poorly understood. Here, we carried out two field experiments to investigate the soil fungal community variations and assembly processes under short- (2 years) versus long-term (13 years) exogenous nitrogen addition (100 kg N ha-1 yr-1) in a N-rich tropical forest of China. Results: We observed that short-term N addition significantly increased fungal taxonomic and phylogenetic α-diversity, and shifted fungal community composition with significant increases in the relative abundance of Ascomycota and saprotrophic fungi, and decreases in the relative abundance of Basidiomycota and ectomycorrhizal (EcM) fungi. However, unremarkable effects were found under long-term N addition. The variations of fungal α-diversity, community composition, the relative abundance of major phyla, genera and functional guilds were mainly correlated with soil pH and the concentrations of NO3--N, and these correlations were much stronger under short- than long-term N addition. The results of null, neutral community models and the 39 normalized stochasticity ratio (NST) index consistently revealed that stochastic processes played predominant roles in the assembly of soil fungal community under N addition in the tropical forest, and that the relative contributions of stochastic processes were higher at short-term site. Furthermore, both short- and long-term N addition slightly loosened the co-occurrence networks of the fungal community. Conclusions: These findings highlighted that the responses of fungal community structure to N addition were duration-dependent, i.e., the fungal community was sensitive to the short-term N addition but become acclimatized to long-term N enrichment.

Plant hydraulic traits have been shown to be sensitive to changes in nitrogen (N) availability in short-term studies largely using seedlings or saplings. The extent and the magnitude of N-sensitivity of the field grown mature trees in long-term experiments, however, are relatively unknown. Here, we investigated responses of leaf water relations and morphological and anatomical traits of two dominant tree species (Castanopsis chinensis and Schima superba) to a six-year canopy N addition in a subtropical forest. We found that N addition increased leaf hydraulic conductivity in both species along with higher transpiration rate and less negative water potential at 50% loss of leaf hydraulic conductivity and at leaf turgor loss point. Examination of leaf morphological and anatomical traits revealed that increased leaf hydraulic efficiency was at least in part due to increased vessel diameter which also compromised the hydraulic safety under increased water stress. Moreover, reduced vessel reinforcement and increased thickness shrinkage index further interpreted the increases in leaf hydraulic vulnerability under N addition. Our results demonstrated that N deposition may lead to increases of plant water loss to the atmosphere as well as tree vulnerability to drought.

We investigated the impacts of 14-year continuous N addition on activities of arylsulfatase (AS) and phosphodiesterase (PDE), which catalyze soil organic sulfur (S) and phosphorus (P), respectively. The response of AS to N addition was compared with that of C- and N-acquiring enzymes, i.e., β-1,4-glucosidase (BG), β-D-cellobiohydrolase (CBH), β-1,4-Xylosidase (BX), β-1,4-N-acetyl-glucosaminnidase (NAG), and leucine amino peptidase (LAP). We also compared the impact of N addition on PDE activity with that on phosphomonoesterase (PME) activity. The results showed that N addition clearly decreased soil AS activity, whereas activities of C- and N-acquiring enzymes did not exhibit similar changes. The inconsistent response of AS activity with the enzymes was attributed to soil acidification induced by long-term N addition, which shifts the pH condition to a more optimal condition for AS activity and accelerates the accumulation of S in soils. The ratio of PME to PDE were significantly elevated by N addition, implying that the microbial and plant resource allocation to PME relative to PDE increased after N addition at our study site. The shift in resource allocation may have occurred because (i) the types of phosphatases secreted by biota shifted toward phosphatases that require one-step reaction to obtain P (such as PME) in the more-severely P-depleted condition, or (ii) soil acidification provided a more optimal condition for PDE than PME, which resulted in lower PDE production appearing as lower enzyme activity at the same pH condition in the laboratory. Overall, our results indicated different responses of exoenzyme activities to external N input. This highlights the importance of atmospheric N deposition on microbial activity and collateral C and nutrient dynamics in tropical natural forests.

The impact of nitrogen (N) deposition on dissolved organic carbon (DOC) fractions in throughfall is not well understood. We performed a laboratory experiment and compared DOC leaching from canopy leaves after dipping leaves in pure water (control) and NH4NO3 solution (N-treatment) for 18 h. Net changes of DOC, NH4+, NO3−, SO42−, K+, Mg2+, Ca2+ and H+ contents after dipping leaves were determined by comparing solutions with and without leaves. We recorded no differences of DOC leaching between control and N-treatment, implying that N deposition had minor impacts on canopy DOC production. This confirmed that previous experiments testing the effects of N addition on DOC dynamics without considering the effects of the canopy reaction successfully described the real situation. We also confirmed the previously-reported canopy exchange process in spite of a high background N deposition at our study site. N-treatment significantly increased base cation leaching, especially K+, and the increase was positively correlated with foliar NH4+ retention. Net leaching of H+ and SO42− was not affected by the N-treatment.

Lowland tropical forests with chronic nitrogen (N) deposition and/or abundant N-fixing organisms are commonly rich in N relative to other nutrients. The tropical N richness introduces a paradoxical relationship in which many tropical forests sustain high rates of asymbiotic N fixation despite the soil N richness and the higher energy cost of N fixation than of soil N uptake. However, the mechanism underlying this phenomenon remains unclear. Our study aims to test this phenomenon and examine potential mechanisms of nutrient concentrations vs. substrate stoichiometry in regulating N fixation using multiple linear regression models. We hypothesized that the rates of asymbiotic N fixation would be low in an N-rich forest under N deposition and substrate stoichiometry would explain the variation in N fixation better than nutrient concentrations. We conducted a chronic N-addition experiment in an N-saturated tropical forest in southern China and measured the N fixation rates, carbon (C), N, and phosphorus (P) concentrations, and stoichiometry in different substrates (soil, forest floor, mosses, and canopy leaves). Total N fixation rates were high (10.35-12.43 kg N·ha-1 ·yr-1 ) in this N-saturated forest because of the high substrate C:N and N:P stoichiometry (which explained 13-52% of the variation in N fixation, P 0.05). Atmospheric N deposition (34-50 kg N·ha-1 ·yr-1 ) failed to down-regulate asymbiotic N fixation in this forest possibly because the N deposition rate was insufficient to inhibit N fixation or N deposition maintained high N fixation rates by increasing C sequestration in the substrates. Our N-addition experiment showed the insensitivity of N fixation in all the tested substrates to low N addition (50 kg N·ha-1 ·yr-1 ); however, medium and high N addition (100-150 kg N·ha-1 ·yr-1 ) stimulated the moss and foliar N fixation because of the increases in substrate C:N stoichiometry (which explained 30-34% of the variation in N fixation, P < 0.001). Overall, our results emphasize the importance of substrate (particularly mosses and foliage) stoichiometry as a driver of asymbiotic N fixation and sustained N richness in lowland tropical forests.