Soil nitrogen (N) transformation, a key microbial process in global N cycling, is thought to alter soil N availability and subsequently regulate ecosystem functioning. In particular, the gross rates of N transformation can provide a deeper understanding of internal N dynamics and mechanisms, but questions of whether gross N transformation processes and their underlying drivers change with seasons and elevations remain large uncertain.

Based on field collection along an elevational gradient and laboratory incubation experiments, we investigated the seasonal processes of soil N mineralization and nitrification with 15N isotope dilution technique, and also explored the potential mechanisms involved in the Hengduan Mountains.

Unimodal soil gross/net mineralization rates were higher in the wet season (61.32 mg kg^-1 d^-1; 1.01 mg kg^-1 d^-1; respectively) than in the dry season (10.88 mg kg^-1 d^-1; 0.55 mg kg^-1 d^-1; respectively) (P < 0.001), with a peak at medium elevation. Soil gross/net nitrification rates were lower in the wet season (1.43 mg kg^-1 d^-1; -0.017 mg kg^-1 d^-1) than in the dry season (2.49 mg kg^-1 d^-1; 0.004 mg kg^-1 d^-1; respectively) (P < 0.001), which increased with increasing elevation. The results detected the dominant drivers of the gross transformation rates, specifically microbial attributes occupied crucial roles in controlling the gross mineralization/nitrification in the wet season, and soil physicochemical properties were dominant controllers on the gross mineralization/nitrification during the dry season.

This study revealed the divergent patterns and drivers of N transformation, suggesting that seasonal N cycling should no longer be overlooked if we are to predict N biogeochemical cycles in response to environmental change accurately.

Three field experiment plots (20×20 m) were established along elevational belts. Five to eight fully expanded upper leaves from different canopy were collected from 10 trees of each of the primarily predominant species in each plot. Then two sub-plots (1×1 m) were selected at two corners of each main plot to collect composite litter samples. All plant samples were oven-dried at 60 ℃ to constant weight in the laboratory. Ten soil samples were taken randomly from each sub-plot of the two layers of topsoil (five for 0 – 10 cm and five for 10 – 20 cm) using a stainless steel cylinder (5-cm diameter) and were thoroughly mixed as two composite samples.

Gross N transformation rates were calculated using the differences in atm% ¹⁵N enrichment and inorganic nitrogen between the pre- and post- incubation. Four sub-samples of 15 g (dry weight basis) of fresh soil were placed into 250 mL flask. The soil samples in flasks were acclimated at 20 ℃ for 7 d, in the dark to allow equilibration. For all flasks, 2 mL of either (¹⁵NH₄)₂SO₄ or K¹⁵NO₃ solution labeled with ¹⁵N at 30 atom% excess was applied to the soil, which is added at an amount equivalent to 20 mg kg^-1 of N. And the final soil moisture contents were adjusted to 60% water holding capacity (WHC) using deionized water. Subsequently, all flasks containing the soil samples were incubated at 20 ℃ for 1 d in the dark. One sub-set of incubation flasks were destructively sampled and extracted using 2 M KCl solution on pre- and post-incubation (T₀ and T₁) for analysis of NH₄⁺-N and NO₃^--N concentrations (Dannenmann et al. 2006). The NH₄⁺-N and NO₃^--N concentrations were quantified with a Cleverchem 380 automatic discrete analyzer (DeChem-Tech. GmnH Inc., Germany). The abundances of ¹⁵N in NH₄⁺-N was diffused into acid filter traps with magnesium oxide for converting NH₄⁺-N to liberated ammonia (NH₃) and then was absorbed by filter paper (acidified with 10 μL of 1 M oxalic acid). Subsequently, the extracts were continuously diffused with acidified filters. Afterwards, the abundances of ¹⁵N in NO₃^--N was diffused again with Devarda’s alloy for transferring NO₃^--N to liberated ammonia NH³, and then was absorbed by new acidified filter paper. The acid filters containing N were analyzed ¹⁵N abundance using an isotope ratio mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, USA) after drying at 60 ℃.

Soil net N transformation rates were calculated using pre- and post-incubation extractable inorganic N. After preincubation, two sub-samples (equivalent to 15 g dry weight) were used to calculate soil net mineralization and nitrification rates. One sub-sample was extracted with 2 M KCl (t₀), the other was extracted with same method after incubation at 20 ℃ for 7 days in the dark (t₁).). The NH₄⁺-N and NO₃^--N concentrations were determined using an Automatic Discrete Analyzer. The activities of two soil enzymes, NAG and LAP, were measured by a colorimetric method to explore their effects on soil mineralization.

We used a MoBio PowerSoil™ DNA isolation kit (MoBio Laboratories Inc., Carlsbad, CA, USA) to extract soil (0.25 g) total genomic DNA according to the instructional methods. The reactions were performed in 15 μL mixture containing 7.5 μL of 2× SYBR Green Mix (Vazyme), 0.7 μL of each primer, 1 μL of DNA template, and double-distilled H₂O. A denaturation step was carried out at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, and 60 °C for 30 s, and final extension at 72 °C for 5 min. The PCR product was separated by electrophoresis and purified using a CFX96 Optical Real-Time Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA).