Contrasting regulating effects of soil available nitrogen, carbon, and critical functional genes on soil N 2 O emissions between two rice-based rotations

Contrasting regulating effects of soil available nitrogen, carbon, and critical functional genes on soil N 2 O emissions between two rice-based rotations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Contrasting regulating effects of soil available nitrogen, carbon, and critical functional genes on soil N 2 O emissions between two rice-based rotations Peng Xu, Mengdie Jiang, Imran Khan, Minghua Zhou, Muhammad Shaaban, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3428312/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims Although the effects of upland and flooded rice cultivation on soil N 2 O emissions have been reported, scholars have not comparatively investigated the mechanism underlying N 2 O emissions during the rice cultivation seasons of rice-based rotation systems. Methods Herein, a two-year field experiment including two rice cultivation modes, namely, conventional upland rice–rapeseed (UR-CC) and flooded rice–rapeseed (RR-CC) rotations, was conducted to determine the effect of different rice plantation models on soil N 2 O emissions. Non-rice treatments (UR-NC and RR-NC) during the rice season were also implemented to confirm the effect of rice plantation or soil condition on N 2 O emissions. Results Seasonal N 2 O emissions were higher in UR-CC rotation than in RR-CC rotation (1.54 ± 0.16 vs . 0.71 ± 0.20 and 2.57 ± 0.28 vs. 0.76 ± 0.04 kg N ha -1 for the first and following rice cultivation seasons, respectively). Also, N 2 O emissions were higher in UR-NC treatment than that in RR-NC treatment during both rice seasons (2.45 ± 0.07 vs . 1.43 ± 0.35 and 3.74 ± 0.37 vs . 1.16 ± 0.08 kg N ha -1 , respectively). The yield-based N 2 O emissions were higher in the UR model than in the RR model (0.21 ± 0.01 vs . 0.10 ± 0.02 and 0.34 ± 0.03 vs. 0.11 ± 0.01, respectively). The responses of N 2 O emission fluxes to soil ammonium (NH 4 + ) and dissolved organic carbon (DOC) in UR rotation were stronger than those in RR rotation. Furthermore, total N 2 O emissions from non-rice treatments were higher than those from rice-cultivated treatments for both rice-based rotations. The increase in N 2 O emissions in UR-NC treatment could be attributed to the higher abundance of amoA gene and elevated soil mineral nitrogen content compared to UR-CC treatment. The higher amount of N 2 O generated in RR-NC treatment than that in RR-CC treatment was ascribed to the increased abundance of the nirS gene and the decreased abundance of the nosZ gene. The structural equation model supported that soil moisture, temperature, available C and N, and ammonium oxidation-related functional genes explained more than 70% of the effect on soil N 2 O emissions in UR rotation. Meanwhile, soil moisture, temperature, available N, and denitrification-related functional genes explained 80% of the effect in RR rotation. Conclusions These findings highlight the importance of rice plantation and their contribution to decreased field N 2 O emission, and suggest that soil available C, N, and critical functional genes should be considered when investigating N 2 O mitigation pathways during rice cultivation seasons. Rice-based rotations N2O emissions Soil variables Regulatory factors Functional genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nitrous oxide (N 2 O), one of the main greenhouse gases in the atmosphere, has attracted considerable attention from academics across the world due to its considerably stronger radiative force than a comparable amount of carbon dioxide over a 100-year time frame (IPCC, 2013; Tian et al., 2020). Agricultural soils are the main source of atmospheric N 2 O and account for 60% of all anthropogenic emissions worldwide (Scheehle et al., 2006; Hu et al., 2015). Most of N 2 O in soil is generated by nitrification and denitrification governed by microbial metabolism (Firestone and Davidson, 1989). Cultivation of rice ( Oryza sativa ) contributes a large amount of atmospheric N 2 O emissions (Xing et al., 2002; Kritee et al., 2018); in this regard, the mitigation of N 2 O emissions from rice-based fields has attracted much attention. Nitrogen (N) fertilization is essential to support crop growth. Rice plants absorb N nutrition during their growth period, thereby affecting physical, chemical, and biological processes that regulate soil N 2 O production and emission (Ruser et al., 2001; Kögel-Knabner et al., 2010). In particular, rice growth competes with microorganisms for active carbon (C) and N in soil, leading to substrate limitation for nitrification and denitrification and reduced soil N 2 O production (Xing et al., 1998; Hodge et al., 2000; Davidson, 2009). However, N fertilizer is usually manually spread by farmers before rice is transplanted; as such, some bare soils are also fertilized, resulting in a pulse N 2 O efflux. For instance, the average soil N 2 O emission rate is lower in paddy fields planted with rice than in fields without rice planted (Kim et al., 2021). The decrease in N 2 O emission from paddy fields with rice cultivation could be attributed to the lower average soil inorganic N during the growth period. High soil inorganic N content in bare soils provides sufficient substrates to produce N 2 O and allow the growth of related microorganisms, thereby promoting N 2 O emission (Xu et al., 2023). However, the positive effect of rice cultivation on soil N 2 O emission has also been documented (Yu et al., 1997; Yan et al., 2000), which is plausible because the consumption of oxygen (O 2 ) in the rice plant rhizosphere generates a favorable condition for denitrification-related N 2 O production (Li et al., 2022). The secretion of organic C sources from rice roots in the form of exudates also provides C substrates for microbial denitrification activities, which may enhance soil N 2 O production and emission. Although changes in the amount of available C and N in soil can significantly regulate N 2 O emissions from paddy fields (Xu et al., 2022a, b, 2023), limited information is available about the effect of rice plantation on soil C and N substrate availability, and microbial metabolism, which consequently affect soil N 2 O emissions under different rice-based rotations (Xu et al., 2022b). Thus, the mechanism through which rice farming affects soil accessible C and N, particularly soil dissolved organic C (DOC), mineral N, and microbial biomass C and N (MBC and MBN), should be investigated. Soil available C and N are pivotal substrates for microbial activities related to N 2 O production and consequent emissions (Kader et al., 2013; Sanchez-Martín et al., 2008; Chen et al., 2021; Zhang et al., 2022). In addition to soil available C and N, a number of studies are available on soil N 2 O emission in response to soil variables, microbial community composition, and key functional genes controlling N 2 O production and consumption (Qin et al., 2018; Kong et al., 2021; Wei et al., 2021; Lin et al., 2023). Ammonium oxidation controlled by ammonia-oxidizing archaea and bacteria (AOA- amoA and AOB- amoA ) is one of the main limiting steps of nitrification (Tokutomi et al., 2010; Zou et al., 2022). Moreover, the conversion of nitrite into nitric oxides by nirS , nirK genes and N 2 O into dinitrogen by nosZ gene are the main limiting steps of denitrification (Zhou et al., 2020; Li et al., 2021). Several studies investigated the responses of these genes and N 2 O emissions to N addition and flooded conditions (Shaaban et al., 2018; Qin et al., 2018; Zhou et al., 2020; Yang et al., 2022). Scholars also emphasized the importance of these key factors when investigating soil N 2 O emissions. Future works should determine whether rice cultivation influences the production and consumption of N 2 O and the abundance of several important functional genes, which could affect soil N 2 O release (Ma et al., 2008; Yang et al., 2017). Rice-based cultivation in China is critical in regulating soil N 2 O emission, and the effect of rice plantation on several key functional genes that regulate N 2 O emission has not been extensively studied across different rice cultivation modes. In this regard, the present study selected two frequently rice cultivation rotation systems, namely, upland rice–rapeseed (UR) and flooded rice–rapeseed (RR), to investigate the effect of rice plantations on soil N 2 O release (during the rice-growing seasons). This study aimed to i) assess the contribution of soil available C and N fractions related to N 2 O emissions (i.e., soil DOC, mineral N, MBC and MBN contents) in two rice-based rotation systems and ii) explore the potential relationship between N 2 O emissions and soil-related C and N fractions and microbial functional genes. We hypothesized that rice plantation would change soil available C and N fractions due to their absorption and utilization of active C secretion, consequently affecting the abundance of key functional genes related to N 2 O production and consumption regardless of rice-based rotations. Thus, several key environmental factors, soil characteristics, and microbial functional genes corresponding to soil N 2 O production and consumption were investigated. Materials And Methods Description of study site and design of field experiments The experimental field in HuangGang Academy of Agricultural Sciences, Hubei Province, China (30°63'N, 114°87'E), was selective to carry out the field trial. Area where the trial site is located has a subtropical monsoon environment with mean yearly temperature and precipitation of 18 °C and 1100 mm, respectively. Table S1 shows the soil physicochemical properties including texture, bulk density (BD) total organic C, total N, and pH at the onset of the study. The experiment was conducted from May 2019 to September 2020, which included two rice plantation seasons (from May 2019 to September 2019 and from May 2020 to September 2020, respectively) and one rapeseed season (from October 2019 to May 2020). Real data on N 2 O emission from January to March 2020 during the rapeseed season were missing due to the epidemic of COVID-19. Two rice cultivation rotation modes, namely, UR and RR, were selected. For the UR model, which means rapeseed–aerobic rice rotation, aerobic rice was cultivated in summer without flooding, while for RR model, which represents rapeseed–flooded rice rotation, paddy rice seedlings were transplanted in late May. For both UR and RR rotations, winter rapeseed seeds were sown in early October. A split-plot experiment was conducted in a plot area of 24 m 2 (4 m × 6 m). Within each plot, a 1.5 m ×1.5 m area was implemented with conventional fertilization but without rice plantation (bare soil with conventional fertilization) to confirm the effect of rice plantation and soil conditions, a part of the non-rice treatments during the rice season. Each treatment was repeated three times. Four treatments were used in total: 1) conventional upland rice–rapeseed rotation (UR-CC); 2) upland rice–rapeseed without rice plantation (UR-NC); (3) conventional flooded rice–rapeseed (RR-CC); and (4) flooded rice–rapeseed without rice plantation (RR-NC). The fertilizer applied during the upland rice season and the flooded rice season consisted of 225 kg N ha -1 , 112 kg P 2 O 5 ha -1 , and 225 kg K 2 O ha -1 . All K and P fertilizer dosages were applied as base fertilizer along with one-half of the N fertilizer dose, and the other half was applied as topdressing. During the rapeseed season, the fertilizer applied included 192 kg N ha -1 , 112 kg P 2 O 5 ha -1 , and 112 kg K 2 O ha -1 . All K and P fertilizers were applied as base fertilizer, and only one component of the N fertilizer (60%) was applied as base fertilizer. The leftover portion of N was applied as topdressing. Table S2 shows detailed information on fertilizer application and management practices for the treatments. N 2 O flux measurement N 2 O gas samples were collected from the treated areas by using static opaque chamber technique (Wang et al., 2013). The chamber was made up of a foundation frame and a chamber lid. The chamber cover, which is covered in insulating foam, has a bottom area of 0.25 m 2 and a height of 1 m, while the base structure has the same bottom area as the chamber and a height of 0.3 m. For gas sampling during the rapeseed growth season, another chamber with the height of 0.5 m was combined with the chamber with the height of 1 m when the rapeseed grew up more than 1 m. After plowing the plot at the start of each growing season, the foundation frame was inserted manually into the soil. An upper edge that can be filled with water was specifically designed for each base to maintain the chamber’s liquid closure prior to gas sampling. Five tubes of gas samples were manually taken from each chamber by using 50 mL syringes, and a 10 min gap was implemented between each sampling event. Gas was collected twice weekly, but the frequency was adjusted to seven times a week following fertilization and heavy rain. Within 24 hours of collection, gas sample concentrations were determined using a gas chromatography (GC; 7890A, Agilent Technologies, California, USA) system fitted with an electron capture detector (ECD). Following the measurement, N 2 O fluxes and seasonal total N 2 O emissions from each plot were calculated using the approaches proposed by Zheng et al. (2008). The data of N 2 O emission fluxes during the first rice-cultivated season in 2019 for RR-CC treatment were obtained from the study of Xu et al. (2023). Auxiliary parameter measurements Other key variables were determined (Xu et al., 2022a). A small digital thermometer was used to capture ambient temperature inside each compartment (JM624, Liwen Electronics Ltd., Tianjin, China). A vertical ruler was used to measure the field inundation water level (only for the rice-planting season in the RR system). Other crucial factors were measured concurrently with the monitoring of N 2 O flow (Xu et al., 2022a). During gas sample collection, the temperature of the soil at a 5 cm depth was manually measured using an electronic probe. Each week, five random topsoil (0-20 cm) samples were collected from each plot and manually blended to produce a single composited soil sample. The sample was quickly transported to the laboratory and stored in a refrigerator at 4 ℃. A portion of soil samples was used to measure soil water content. Another part of soil samples was used to determine soil pH, available C and N contents suggested by Xu et al. (2022b). Another part of the soil samples was used to determine soil microbial biomass C and N (MBC and MBN) by chloroform fumigation method, which was proposed and modified by Brookes et al. (1987). Soil bulk density (BD) was determined using columnar soil samples collected using cylinder rings at the beginning of the experiment. A modest auto-monitoring meteorological station located close to the experimental location was used to record air temperature and precipitation daily. Water filled pore space (WFPS), which describes the soil moisture during the crop-growing period for the UR system, was computed by the following equation: where 2.65 g cm −3 was assumed as soil particle density (PD). The partial data of soil parameters during the first rice-planting season in 2019 for RR-CC treatment were obtained from the study of Xu et al. (2023). Real-time quantitative PCR (qPCR) The abundance of several key functional genes involved in N 2 O production and consumption ( amoA , nirS , nirK , and nosZ genes) was quantitatively determined by real-time quantitative PCR procedure (qPCR) using the referenced primer sets (Table S3). Approximately 0.35 g of fresh soil was sifted through a 2 mm sieve and used to extract soil microbial DNA with Fast DNA SPIN Kit (MP Biomedicals, USA) based on the manufacturer’s instructions. After microbial DNA extraction, qPCR analysis was performed by SYBR Green method. The measurement range and the R 2 value of each calibration curve were set at 95% to 110% and higher than 0.98, respectively. The total volume of 15 µL of the quantitative PCR reaction liquid was composed of 7.5 µL of 2×SYBR Green Mix (TaKaRA Japan), 1 µL of diluted template DNA, and 0.7 µL of primer, and the remaining volume was sterile water. The amplification conditions described in a previous study (Xu et al., 2022b) was referenced to DNA amplification. The formulae mentioned by Macdonald et al. (2011) were used to calculate the copy number of each gene. Statistical analyses Data displaying normality were used directly, while data that had non-normality were analyzed and log-transformed before use. Cumulative seasonal N 2 O emissions were compared among the four treatments by using the analysis of variance (ANOVA) procedure. Functional relationships among N 2 O emission fluxes and soil parameters, and N 2 O production and consumption-related functional genes were described using linear or nonlinear regression. Structural equation models (SEM) were used to exhibit the direct and indirect effects of aforementioned factors on N 2 O emissions by using AMOS software (AMOS 21.0, SPSS Inc., Chicago, USA). Additionally, SPSS (version 19.0, SPSS Inc., Chicago, USA) and Origin 8.0 tools (Origin Lab Corporation, USA) were used for figure creation and statistical analysis. Finally, a p ≤ 0.05 level was set to indicate a significantly statistical difference. Results Climate, environmental, and soil variables The daily air and soil temperatures at 5 cm depth ranged from -3.3 °C to 33.6 °C and from 4.8 °C to 32.7 °C, respectively, over the experimental period. The recorded annual precipitation levels were 1103.9 and 559.3 mm during the 2019–2020 period and the following rice season in 2020, respectively (Fig. S1). During the first and following upland rice-planting seasons, soil WFPS varied from 35.6 to 68.8% and 37.4 to 64.7% in the UR-CC treatment and 34.1 to 69.2% and 36.7 to 62.9% in the UR-NC treatment, respectively (Fig. S1). During the first and following flooded rice-planting seasons, the flood water depths were 0–4.65 and 0–4.72 cm in the RR-CC treatment and 0–4.55 and 0–4.62 cm in the RR-NC treatment, respectively (Fig. S2). In the initial and succeeding upland rice cultivation seasons, the soil DOC ranged from 20 mg C kg -1 to 50 mg C kg -1 for the UR-CC and UR-NC treatments. For the RR-CC and RR-NC treatments, the soil DOC ranged from 20 mg C kg -1 to 60 mg C kg -1 during both flooded rice-planting seasons (Fig. 1a). The average soil DOC content in the UR rotation system was comparable with that in the RR rotation system (Table S4). For all treatments, the soil NH 4 + -N content increased sharply with basal N fertilization and subsequently decreased to a low level. The lowest soil NH 4 + -N content was approximately 4 mg N kg -1 , while the highest value was more than 90 mg N kg -1 during the upland rice cultivation season in UR rotation. The corresponding lowest value was approximately 1 mg N kg -1 , and the highest value was more than 65 mg N kg -1 during the flooded rice cultivation seasons in RR rotation. The average soil NH 4 + -N content was higher in the UR rotation system than in the RR rotation system (Fig. 1b). The soil NO 3 - -N content increased with basal N fertilization and subsequently decreased to a low level (Fig. 1c). The average soil NO 3 - -N contents were significantly higher in UR rotation than in RR rotation (Table S4). For all treatments, soil MBC content dynamics exhibited similar patterns for the two upland rice growing seasons as well as for the two flooded rice growing seasons. The highest average soil MBC content was detected in the UR-CC treatment, with ranges of 74.91–638.20 and 160.38– 625.07 mg C kg -1 during the first and following upland rice cultivation seasons, respectively (Fig. 2a). Meanwhile, the highest average soil MBN content was observed in the UR-CC treatment, with ranges of 20.21–46.40 and 13.42–45.35 mg N kg -1 during the first and following upland rice cultivation seasons, respectively (Fig. 2b). The MBC/MBN ratio was higher in RR rotation than in UR rotation (Fig. 2c). N 2 O emissions in the UR-CC treatment, the N 2 O emission fluxes were -8.89–644.59 and 1.92–1536.22 μg N m -2 h -1 during the first and following upland rice-planting seasons, respectively. Higher N 2 O emission fluxes of -1.13–1196.43 and -19.32–1607.49 μg N m -2 h -1 were observed in the UR-NC treatment compared with those in the UR-CC treatment. In RR rotation, the N 2 O emission fluxes were -4.06–564.65 and -18.12–803.22 μg N m -2 h -1 in the RR-CC treatment during the flooded rice-planting seasons. Higher N 2 O emission fluxes were observed in the RR-NC treatment compared with those in the RR-CC treatment, with ranges of 3.68–664.65 and 3.65–721.02 μg N m -2 h -1 , respectively (Fig. 3). The seasonal cumulative N 2 O emissions in the UR-CC treatment were 1.54 ± 0.16 and 2.57 ± 0.28 kg N ha -1 for the first and second upland rice-planting seasons, respectively, which were significantly lower than those in the UR-NC treatment, which had total N 2 O emissions of 2.45 ± 0.07 and 3.74 ± 0.37 kg N ha -1 , respectively (embedded graph in Fig. 3). However, compared with UR rotation, RR rotation had lower seasonal cumulative N 2 O emissions of 0.71 ± 0.20 and 0.76 ± 0.04 kg N ha -1 in the RR-CC treatment and 1.43 ± 0.35 and 1.16 ± 0.08 kg N ha -1 in the RR-NC treatment during the first and following flooded rice-planting seasons, respectively (embedded graph in Fig. 3). Abundance of AOA-amoA, AOB-amoA, nirK, nirS, and nosZ genes For UR and RR rotations, the abundance of the AOB- amoA gene was approximately two orders of magnitude lower than that of the AOA- amoA gene regardless of the rice planting season (Fig. 4). The average copy number of the AOA- amoA gene during the upland rice or flooded rice cultivation seasons for the rice planting treatments was significantly lower than those for the non-rice planting treatments (Fig. 4a). The average copy number of the AOB- amoA gene during the upland rice cultivation seasons for the UR-CC treatment was significantly lower than that for the UR-NC treatment (Fig. 4b). The average copy number of the AOB- amoA gene in the RR-CC treatment during the flooded rice-growing seasons was higher than that in the RR-NC treatment (Table S5). In particular, the gene copy number ratio of the AOA- amoA to AOB- amoA (AOA/AOB) was significantly higher in UR rotation than in RR rotation (Fig. 4c, Table S5). The average nirK gene copy number in UR rotation during the upland rice-growing seasons was significantly lower than those in RR rotation during both flooded rice-growing seasons (Fig. 4e, Table S6). The average copy number of the nirS genes in the RR-CC treatmentduring the flooded rice-growing seasons were significantly lower than that in the RR-NC treatment (Fig. 4d, Table S6). For upland rice-growing seasons, the average copy number of the nirS and nirK genes was comparable between the rice-planted and non-rice treatments under UR rotation (Fig. 4d, e, Table S6). For RR rotation, the average copy number of the nosZ gene was higher in rice-planted treatments than in non-rice-planted treatments (Fig. 4f, Table S6). In UR and RR rotations, the value of nosZ /( nirS + nirK ) was higher in rice-planted treatments than in non-rice-planted treatments (Fig. 4g, Table S6). Relationship between N 2 Oemissions and soil parameters and functional genes According to Fig. 5, the soil N 2 O flux increased linearly with increasing soil NH 4 + and DOC concentrations in the RR-CC and RR-NC treatments. The N 2 O fluxes in soils treated with UR-CC and UR-NC exponentially increased with increasing soil DOCcontent (Fig. 5a) and linearly increased with increasing soil NH 4 + level (Fig. 5b). Logarithmic relationships were observed between N 2 O fluxes and the value of DOC/NO 3 − for various treatments during the rice-growing seasons (Fig. 5c). Furthermore, positive linear relationships between N 2 O fluxes and the value of AOA /AOB were observed in UR rotation (Fig. 5d). In RR rotation, N 2 O fluxes were positively linearly related to the copy number of the nirS gene (Fig. 5e) but negatively linearly related to the copy number of the nosZ gene (Fig. 5f). SEM describing the effect of key factors on N 2 O emissions As shown in Fig. 6, an SEM was applied to describe the direct and indirect effects of several key components on soil N 2 O emissions. In particular, soil moisture and/or soil temperature, soil C and N availability, and functional genes (AOA- amoA, AOB- amoA ,and their ratio) explained 76% and 81% of soil N 2 O emissions in the UR-CC and UR-NC treatments, respectively (Fig. 6a and b). Meanwhile, soil temperature and soil moisture, soil C and N availability, and functional genes ( nirS and nosZ ) explained 79% and 83% of soil N 2 O emissions in the RR-CC and RR-NC treatments, respectively (Fig. 6c and d). Yields from different crop seasons Table S7 shows that the rapeseed yield had no significant difference between the UR-CC and RR-CC treatments throughout the rapeseed-growing season, with yields amounting to 5.67 ± 0.66 and 5.95 ± 0.60 t ha -1 , respectively. The rice yields in the UR-CC treatment during the first and following rice-growing seasons were 7.36 ± 0.48 and 7.58 ± 0.29 t ha -1 , respectively, which were higher than those in the RR-CC treatment (6.80 ± 0.54 and 6.72 ± 0.69 t ha -1 , respectively). Furthermore, higher response of N 2 O emissions to per unit rice yield was observed in the UR-CC treatment than that in the RR-CC treatment for both rice-growing seasons (0.21 ± 0.01 vs .0.10 ± 0.02 and 0.34 ± 0.03 vs . 0.11 ± 0.01, respectively). Discussion Effects of rice-based rotation on soil N 2 O emissions Different cropping patterns have a significant effect on N 2 O emissions from rice fields (Zhou et al., 2017; Zhou et al., 2022b; Cheng et al., 2022; Xu et al., 2023). Regardless of rice planting, N 2 O emissions from the tested soils were higher during the upland rice-growing seasons than during the flooded rice-growing seasons. A field trial revealed significantly higher seasonal N 2 O emissions from cultivated aerobic rice soil than that from soils planted with flooded rice (Mohanty et al., 2017). These differences could be attributed to the large variations in field water management between upland rice and flooded rice cultivation, which further influenced soil N 2 O emissions (Liu et al., 2010; Shaaban et al., 2018). In the present study, the moisture content (WFPS) of upland paddy soil was 45%–60%, which is conducive to nitrification by soil microorganisms, thereby promoting soil N 2 O emission (Qin et al., 2018). The highest soil N 2 O emission occurred when the soil WFPS ranged from 45% to 75%, where nitrifying microorganisms had vigorous activities (Sánchez-Martín et al., 2008). In the UR system, soil N 2 O emission fluxes during the rice season were related to the AOA/AOB ratio in the UR-CC and UR-NC treatments, indicating that soil N 2 O emissions were mainly contributed by nitrification dominated by AOA. Wang et al. (2021) reported that although organic material amendments were added to upland soil, N 2 O loss derived from denitrification was negligible. In addition, the depth of the water layer during the rice-growing season is an important factor affecting soil N 2 O emissions (Xu et al., 2022a, b). Regardless of drainage during the mid-season and after maturity, the paddy field typically remained flooded during the rice season in RR mode, which was conducive to the progress of denitrification (Di et al., 2014). Prolonged flooding promoted complete denitrification, indicating the conversion of N 2 O into N 2 . This finding was confirmed by the relationship between N 2 O and denitrifying functional gene nosZ (Fig. 5f). In contrast, higher inorganic N levels were observed in UR mode than in RR mode, which may have provided a sufficient substrate for nitrifying and denitrifying microbial activities. Our study revealed that inorganic N affected functional genes related to nitrification, thereby affecting N 2 O production (Fig. 6). Moreover, the average soil DOC content during the rice season was higher in RR rotation than in UR rotation, regardless of rice plantation. Previous investigations suggested soil DOC as an active substrate that can be utilized by microorganisms effectively and thus influenced N 2 O production and emissions (Sanchez-Martin et al., 2008; Shaaban et al., 2019). For example, a higher DOC content can promote soil denitrification to regulate N 2 O emissions (Lee et al., 2017; Zhou et al., 2017b). Apart from the RR-NC treatment, N 2 O emission rates during the rice-growing seasons had a positive association with the value of soil DOC/NO 3 − (Fig. 5c), consistent with the results of previous studies (Hu et al., 2015; Zhou et al., 2017b). Further, Lan et al. (2017) discovered that the value of DOC/NO 3 − should be between 0.69 and 0.84 for the N 2 O emission peak to occur. Zhou et al. (2017b) reported that N 2 O fluxes had a curvilinear relationship with soil DOC/(NH 4 + +NO 3 - ). These results illustrate that the ratio of soil labile C to mineral N can regulate N 2 O emissions from paddy fields. The differences in physiology between upland rice and flooded rice soils could be attributed to their different cropping patterns (sowing vs . transplantation). For instance, compared with flooded rice, upland rice had a higher planting density but considerably smaller roots and stems. Moreover, upland rice had a higher grain yield than flooded rice (Table S7). These differences may further contribute to different nutrient utilization patterns in the studied soils during the two rice-growing seasons, thereby affecting soil C and N cycling and N 2 O production and consumption (Mohanty et al., 2017; Zhang et al., 2022; Xu et al., 2023). In the RR-CC treatment, upland rice with finer roots and higher planting density and grain yield required more mineral N, which might have competed with N 2 O production activities for the common N substrate (Kim et al., 2021). However, in the UR-CC treatment, enhanced N mineralization during upland rice planting period might be enhanced by the following pathways: (1) higher soil DIN and MBN used as substrates by N 2 O production-related microbes and (2) optimum soil aeration conditions in comparison with that during the flooded rice-plantation period. Compared with flooded padded soils, upland rice may have higher grain yield and substantial organic N mineralization, which contribute to greater N 2 O emissions. In UR rotation, higher AOA -amoA gene abundance was found, however, N 2 O flux was not significantly related to the abundance of the AOA- amoA gene but was highly correlated with that of the AOB -amoA gene (Fig. 6) and/or AOA/AOB (Fig. 5d and 6). Moreover, AOB- amoA gene abundance and AOA/AOB were indirectly correlated with N 2 O flux by the middle bridge of soil mineral N contents. In particular, NO 3 − -N was strongly and positively correlated with the AOB- amoA gene, which was associated with N 2 O emission (Fig. 6a). Our results indicate that AOB may play a great role in the microbial release of N 2 O during upland rice growing seasons with lower soil moisture, consistent with the previous findings that AOB may be the primary producers of N 2 O in alkaline pH soils (Zou et al. 2022). Pan et al. (2016) found that AOB plays a dominant role in the microbial release of N 2 O in grassland soils. In RR rotation, the N 2 O emission flux showed a positive correlation with the denitrifying functional gene nirS , consistent with previous experiments (Wang et al., 2022). The nirS gene has a stronger effect on N 2 O production and emission in soil than the nirK gene (Qin et al., 2020; Zhou et al., 2020). However, other works reported that the higher abundance of the nirK gene in soil under flooded rice growing periods may contribute to greater N 2 O emissions. Yang et al. (2022) found the high abundance of the nirK gene and the accompanied greater N 2 O emissions. Basing on these results, we suggest that the role of nirS and nirK genes in N 2 O emissions needs to be further explored in paddy fields in future research. According to Chen et al. (2020), the ( nirS + nirK )/ nosZ value denotes the genetic capacity of denitrifying genes that controls whether N 2 O is converted into N 2 or released into the environment as a greenhouse gas. In UR and RR rotation systems, the value of ( nirS + nirK )/ nosZ was higher in non-rice-planted treatments than in rice-planted treatments, which may further explain the greater N 2 O emissions in the former. In agreement with our results, previous studies attributed the higher N 2 O emissions to higher ( nirS + nirK )/ nosZ (Kong et al., 2021; You et al. 2022). Thus, the ratio of ( nirS + nirK )/ nosZ should be considered when investigating soil N 2 O emission in rice-based field systems in the future. Effects of rice planting on soil N 2 O emission In this study, N 2 O emissions in UR and RR rotation systems were significantly greater in the non-rice planting treatment than in the rice planting treatment, consistent with previous findings (Kim et al., 2021). The average seasonal N 2 O emission rate from paddy soil with plants was lower than that without plants (López-Fernández et al., 2007) because of lower denitrification potential (Kim et al., 2021). Rice growth was presumed to compete for N substrate, which further supported nitrifying and denitrifying microorganisms after rice transplanting. Therefore, non-rice-planted soil with high N might provide sufficient substrates for N 2 O production in microorganisms and enhanced N 2 O emissions from the soil (Opez-Fernande, 2007; Davidson, 2009). The average soil NH 4 + -N concentration was significantly higher in the non-rice treatment than in the crop-planting treatment, providing active substrate and supporting the processes related to N 2 O production. In agreement with our findings, Hodge et al. (2000) discovered that inorganic N concentration was higher in bare soil than in soil cultivated with crops, and it offered a sufficient substrate for nitrifying and denitrifying microorganisms and promoted the synthesis and emission of N 2 O from the soil (Xing et al., 1998; Opez-Fernande, 2007; Zhang et al., 2016). The average MBC and MBN contents in the soil under cropping were higher than those in non-cropping soil, indicating that rice cultivation increased microbial activity, which may promote N 2 O production to some extent. Exudation from rice roots as an organic C sources (Zhu et al., 2018, Khan et al., 2019) provide remarkable energy for microbial denitrification and facilitate N 2 O reduction. However, the negative effect of rice plantation on N 2 O emissions could be expected due to O 2 consumption and favored denitrification during flooded rice-growing period, leading to the reduction of N 2 O into N 2 (Yang et al., 2021). In a double rice-fallow cultivation mode, Xu et al. (2022b) observed that N 2 O emissions were not different between rice-plantation and non-rice plantation treatments. Rice cropping affects functional genes related to nitrification and denitrification, thereby affecting N 2 O emissions (Ma et al., 2008; Yang et al., 2017). The average copy number of the AOA- amoA gene in soil was higher in the non-rice treatments than in the rice plantation treatments in the UR and RR rotation systems. The copy number of the AOA- amoA gene was higher by two orders of magnitude than that of the AOB- amoA gene in UR and RR rotations, highlighting the ecological niche of AOA that occupied the studied soils. Previous studies reported that AOA dominated the paddy soils compared with AOB (Chen et al., 2008). The nosZ gene in soil in the RR-CC treatment was higher than that in the RR-NC treatment. The higher abundance of the nosZ gene may lead to higher nitrous oxide reductase, which modulates the reduction of N 2 O into N 2 , consequently reducing N 2 O emissions (Liu et al., 2010; Xu et al., 2022b). As proposed previously, the prevalence of nosZ -containing microorganisms may be related to the ambient N 2 O concentration because N 2 O serves as the electron receptor for nitrous oxide reductase (Qin et al., 2020). Rice plantations enhanced N 2 O emissions mainly by increasing the abundance of the AOB -amoA and/or the value of AOA/AOB in a typical RR rotation system. The increase in N 2 O emissions could be due to the increasing abundance of the nirS gene and the reducing abundanceofthe nosZ gene in a typical flooded RR rotation system. Implications for future studies Rice cultivation has a significant effect on soil N 2 O emissions. In theory, our findings showed that functional genes related to nitrification and denitrification interacted with important soil factors to govern N 2 O emission. However, soil N 2 O production and consumption may co-occur in rice-based fields that are characterized by changing redox conditions, substrates, and microbial conditions during the rice-growing period. We propose that certain significant environmental variables, soil characteristics, and microbes regulating N 2 O production and consumption will work together to control field-level release of soil N 2 O. As a result, studies should focus on variations in available C and N substrates as well as microorganisms associated with N 2 O generation, which could be affected by the changing soil condition and habitat in a rice-based field. In summary, rice planting could be beneficial to the mitigation of soil N 2 O emissions compared with soil without rice planted but spread with N fertilizer unintentionally. Future studies should investigate soil N 2 O emissions in paddy fields in terms of differences in the characteristics of N 2 O production-related microorganisms and functional genes. For instance, the microbial effect on N 2 O emissions could be further investigated through RNA-based microbial profiling (Wei et al. 2021). Moreover, 15 N- 18 O labeling approach can be used to resolve the quantity of microbial denitrification processes related to N 2 O production, similar to conventional denitrification and nitrifier denitrification (Kool et al. 2010). Conclusion This study examined N 2 O emissions in soil in two different rice cultivation modes, namely, rice planting and non-rice planting treatments. N 2 O emissions in the upland rice seasons were significantly higher than those in the flooded rice cultivation seasons, regardless of rice planting. Although higher rice yield was determined in UR rotation than in RR rotation, the yield-based N 2 O emissions were higher in the UR model than in the RR model. These results could be attributed to the stronger responses of N 2 O emission fluxes to soil available N in UR rotation than in RR rotation. The responses of N 2 O emission fluxes to soil ammonium NH 4 + and DOC in UR rotation were stronger than those in RR rotation. Furthermore, N 2 O emissions from rice-cultivated treatments were higher than those from non-rice treatments for both rice-based rotations. The increase in N 2 O emissions in rice-free treatment compared to rice-cultivated treatment could be attributed to the abundance of AOA- amoA and AOB- amoA genes and elevated soil mineral nitrogen content under UR rotation. While, the higher amount of N 2 O from rice-free treatment compared to rice-cultivated treatment under RR rotation was ascribed to the increased abundance of the nirS gene and the decreased abundance of the nosZ gene. Our results highlight that the response of N 2 O emissions mainly relies on soil available N and C and critical functional genes, which should be the focus when investigating N 2 O mitigation measures in the future. The contribution of rice plantation to decreased field N 2 O emission during rice cultivation seasons should also be investigated in comparison with non-rice plantation. Declarations Acknowledgments Lastly, the writers would like to express their gratitude to Key Research and Development Project of Hubei Province for funding this study (2021BCA156). Conflict of Interest The authors declare no conflict of interest in this paper. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3428312","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":239499399,"identity":"c45c8e98-fd11-49a5-bcd2-e9c15176fc9f","order_by":0,"name":"Peng Xu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Xu","suffix":""},{"id":239499400,"identity":"8181f24f-418b-483c-b713-0cf790e91417","order_by":1,"name":"Mengdie Jiang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mengdie","middleName":"","lastName":"Jiang","suffix":""},{"id":239499401,"identity":"bbc6f3b9-a5a9-49ad-b519-e8d17740cf5b","order_by":2,"name":"Imran Khan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Imran","middleName":"","lastName":"Khan","suffix":""},{"id":239499402,"identity":"9461f51b-d2bd-452b-8d47-c17704727778","order_by":3,"name":"Minghua Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Minghua","middleName":"","lastName":"Zhou","suffix":""},{"id":239499403,"identity":"bed8bbc1-e063-4474-be19-7c21688b8f05","order_by":4,"name":"Muhammad Shaaban","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Shaaban","suffix":""},{"id":239499404,"identity":"cd096a0b-e093-4c0c-85b6-a2fb16716cce","order_by":5,"name":"Ronggui Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYFACHgaGD0CSD8QAgwNEaGGcASTZSNLCDFJMvBbz/rXHpG1z7GTYGHgPf/jZxiDHdyOB8XMBHi0yN96lSeduSwY6jC/BsLeNwVjyRgKz9Aw8WiQkzpgBtTCD/GKQwNvGkLjhRgIbMw8hLZbb6sFaDv5tY6gnrIW/x0yacdthkBbDZqAtCQaEbeExtuzddpwHqMyYWeachOHMMw+bpfHbcsbwxs9t1fb87D3GH9+U2cjzHU8++BmfFgaJBCiDGcIFYsYGfBoYGPgP4JcfBaNgFIyCUcAAANIdPShGtjkFAAAAAElFTkSuQmCC","orcid":"","institution":"College of Resources and Environment, Huazhong Agriculture University, Wuhan 430070, PR China","correspondingAuthor":true,"prefix":"","firstName":"Ronggui","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2023-10-10 16:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3428312/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3428312/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44712027,"identity":"1dfc5d64-0101-4402-ab6d-3a98758295bb","added_by":"auto","created_at":"2023-10-16 17:37:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64398,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics in soil ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, fig. a), nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003efig. b) and dissolved organic carbon (DOC, fig. c) contents for various treatments during the experiment period. Rice plantation and non-rice plantation treatments are represented by the abbreviations UR-CC and UR-NC, respectively, under the upland rice-rapeseed cycle, and RR-CC and RR-NC, respectively, under the flooded rice-rapeseed rotation.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/edd7f9c8eb803c007536a8d1.png"},{"id":44712028,"identity":"f22007d9-23ff-4c70-b502-615b900a5905","added_by":"auto","created_at":"2023-10-16 17:37:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":218973,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics in soil microbial biomass carbon (MBC, fig. a) and microbial biomass nitrogen (MBN, fig. v) contents and their ratio (MBC/MBN, fig. c) for different treatments during the experiment period. Rice plantation and non-rice plantation treatments are represented by the abbreviations UR-CC and UR-NC, respectively, under the upland rice-rapeseed cycle, and RR-CC and RR-NC, respectively, under the flooded rice-rapeseed rotation.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/5a92e29741e10f9c235cab96.png"},{"id":44713154,"identity":"18f092e8-905b-43a1-bf7a-1d7da4a59541","added_by":"auto","created_at":"2023-10-16 17:45:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59631,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal N\u003csub\u003e2\u003c/sub\u003eO emission fluxes and cumulative seasonal N\u003csub\u003e2\u003c/sub\u003eO emissions (embedded figure) for various treatments during the experiment period. Note：Different lowercase over the columns means significant difference between the treatments (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). The red and black arrows denote applying basal nitrogen fertilizer and nitrogen dressing, respectively. Rice plantation and non-rice plantation treatments are represented by the abbreviations UR-CC and UR-NC, respectively, under the upland rice-rapeseed cycle, and RR-CC and RR-NC, respectively, under the flooded rice-rapeseed rotation.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/388b89d9921b23fcb55b2fd2.png"},{"id":44711033,"identity":"57d750e5-9402-428f-b940-56f44a51d22e","added_by":"auto","created_at":"2023-10-16 17:29:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":242603,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics in the abundances of nitrification functional genes (fig. a and b represents AOA\u003cem\u003e-amoA\u003c/em\u003e and AOB\u003cem\u003e-amoA, \u003c/em\u003erespectively) and the ratio of AOA to AOB (fig. c) and in the abundances of denitrification functional genes (fig. d, e and f represents\u003cem\u003e nir\u003c/em\u003eK, \u003cem\u003enirS\u003c/em\u003e and \u003cem\u003enosZ, \u003c/em\u003erespectively) and their ratio (fig. g) for different treatments during the rice-growing seasons.\u003cstrong\u003e \u003c/strong\u003eRice plantation and non-rice plantation treatments are represented by the abbreviations UR-CC and UR-NC, respectively, under the upland rice-rapeseed cycle, and RR-CC and RR-NC, respectively, under the flooded rice-rapeseed rotation.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/dd396fd85482f2459a956824.png"},{"id":44711034,"identity":"2d277cc9-053a-449e-9843-f82ffa034948","added_by":"auto","created_at":"2023-10-16 17:29:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":499659,"visible":true,"origin":"","legend":"\u003cp\u003eRelationships between N\u003csub\u003e2\u003c/sub\u003eO fluxes and soil DOC content (fig. a), NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e content (fig. b), the value of DOC/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e (fig. c), the value of AOA/AOB (fig. d) and the abundances of \u003cem\u003enirS\u003c/em\u003e (fig. e) and \u003cem\u003enosZ\u003c/em\u003e (fig. f) genes for various treatments during the rice-growing seasons. Rice plantation and non-rice plantation treatments are represented by the abbreviations UR-CC and UR-NC, respectively, under the upland rice-rapeseed cycle, and RR-CC and RR-NC, respectively, under the flooded rice-rapeseed rotation.\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/16cfcc5f464ec17fd6b879dc.png"},{"id":44711036,"identity":"75401e9f-7bc1-4b25-809c-ed438672a5f1","added_by":"auto","created_at":"2023-10-16 17:29:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":335956,"visible":true,"origin":"","legend":"\u003cp\u003eStructural equation model (SEM) describing the impact effect of the key factors and functional genes on N\u003csub\u003e2\u003c/sub\u003eO emissions during the rice-growing seasons for various treatments under two rice-based rotations. Soil DOC, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, MBN, WFPS, T, D, AOA and AOB, and \u003cem\u003enirS\u003c/em\u003e and \u003cem\u003enosZ\u003c/em\u003e represents soil dissolved organic carbon (DOC), NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, microbial biomass nitrogen, water filled pore space, soil temperature at 5-cm depth, field flood depth, AOA-\u003cem\u003eamoA \u003c/em\u003eand AOB-\u003cem\u003eamoA\u003c/em\u003e gene abundances and \u003cem\u003enirS\u003c/em\u003e and \u003cem\u003enosZ\u003c/em\u003e gene abundances, respectively. Continuous or dashed lines indicate positive or negative effects, respectively. Width of the arrow indicates strength of the effect; numbers close to lines are standardized direct influence coefficients and R\u003csup\u003e2\u003c/sup\u003e stands for the proportion of variables explained by these drivers. χ2 and \u003cem\u003edf \u003c/em\u003estand for Chi-square and degrees of freedom, respectively; CFI and RMSEA denote comparative fit index and for root mean square error of approximation, respectively; \u003cem\u003ep\u003c/em\u003e represents probability level.\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/f9e1e2fddaa68465f017b3ac.png"},{"id":46746032,"identity":"0913cc85-7f90-44a4-9975-099ab592fcc2","added_by":"auto","created_at":"2023-11-20 04:29:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1614017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/603869b5-d840-49f7-b205-4a96bf0d2371.pdf"},{"id":44711041,"identity":"96817013-cd13-412f-be3e-d144fb5376de","added_by":"auto","created_at":"2023-10-16 17:29:38","extension":"doc","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":127488,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.doc","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/cdda7ac694282ad14bedd2c9.doc"},{"id":44711039,"identity":"6b5baa88-b1a7-4181-9395-94ae426e5d13","added_by":"auto","created_at":"2023-10-16 17:29:38","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":18258,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/8409ef853225bb9172551edb.docx"},{"id":44711044,"identity":"65a886dc-ee58-4eb0-832f-ff23b8b83188","added_by":"auto","created_at":"2023-10-16 17:29:39","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":94173,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3428312/v1/5a0833765ce079a119400369.docx"}],"financialInterests":"","formattedTitle":"Contrasting regulating effects of soil available nitrogen, carbon, and critical functional genes on soil N 2 O emissions between two rice-based rotations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO), one of the main greenhouse gases in the atmosphere, has attracted considerable attention from academics across the world due to its considerably stronger radiative force than a comparable amount of carbon dioxide over a 100-year time frame (IPCC, 2013; Tian et al., 2020).\u0026nbsp;Agricultural soils are the main source of atmospheric N\u003csub\u003e2\u003c/sub\u003eO and account for 60% of all anthropogenic emissions worldwide (Scheehle et al., 2006; Hu et al., 2015). Most of N\u003csub\u003e2\u003c/sub\u003eO in soil is generated by nitrification and denitrification governed by microbial metabolism (Firestone and Davidson, 1989).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCultivation of rice (\u003cem\u003eOryza sativa\u003c/em\u003e) contributes a large amount of atmospheric N\u003csub\u003e2\u003c/sub\u003eO emissions (Xing et al., 2002;\u0026nbsp;Kritee\u0026nbsp;et al., 2018); in this regard, the mitigation of N\u003csub\u003e2\u003c/sub\u003eO emissions from rice-based fields has attracted much attention. Nitrogen (N) fertilization is essential to support crop growth. Rice plants absorb N nutrition during their growth period, thereby affecting physical, chemical, and biological processes that regulate soil N\u003csub\u003e2\u003c/sub\u003eO production and emission (Ruser et al., 2001;\u0026nbsp;K\u0026ouml;gel-Knabner\u0026nbsp;et al., 2010). In particular, rice growth competes with microorganisms for active carbon (C) and N in soil, leading to substrate limitation for nitrification and denitrification and reduced soil N\u003csub\u003e2\u003c/sub\u003eO production (Xing et al., 1998; Hodge et al., 2000; Davidson, 2009).\u0026nbsp;However, N fertilizer is usually manually spread by farmers before rice is transplanted; as such, some bare soils are also fertilized, resulting in a pulse N\u003csub\u003e2\u003c/sub\u003eO efflux. For instance, the average soil N\u003csub\u003e2\u003c/sub\u003eO emission rate is lower in paddy fields planted with rice than in fields without rice planted (Kim et al., 2021). The decrease in N\u003csub\u003e2\u003c/sub\u003eO emission from paddy fields with rice cultivation could be attributed to the lower average soil inorganic N during the growth period. High soil inorganic N content in bare soils provides sufficient substrates to produce N\u003csub\u003e2\u003c/sub\u003eO and allow the growth of related microorganisms, thereby promoting N\u003csub\u003e2\u003c/sub\u003eO emission (Xu et al., 2023). However, the positive effect of rice cultivation on soil N\u003csub\u003e2\u003c/sub\u003eO emission has also been documented (Yu et al., 1997; Yan et al., 2000), which is plausible because the consumption of oxygen (O\u003csub\u003e2\u003c/sub\u003e) in the rice plant rhizosphere generates a favorable condition for denitrification-related N\u003csub\u003e2\u003c/sub\u003eO production (Li et al., 2022). The secretion of organic C sources from rice roots in the form of exudates also provides C substrates for microbial denitrification activities, which may enhance soil N\u003csub\u003e2\u003c/sub\u003eO production and emission. Although changes in the amount of available C and N in soil can significantly regulate N\u003csub\u003e2\u003c/sub\u003eO emissions from paddy fields (Xu et al., 2022a, b, 2023), limited information is available about the effect of rice plantation on soil C and N substrate availability, and microbial metabolism, which consequently affect soil N\u003csub\u003e2\u003c/sub\u003eO emissions under different rice-based rotations (Xu et al., 2022b). Thus, the mechanism through which rice farming affects soil accessible C and N, particularly soil dissolved organic C (DOC), mineral N, and microbial biomass C and N (MBC and MBN), should be investigated. Soil available C and N are pivotal substrates for microbial activities related to N\u003csub\u003e2\u003c/sub\u003eO production and consequent emissions (Kader et al., 2013; Sanchez-Mart\u0026iacute;n et al., 2008; Chen et al., 2021; Zhang et al., 2022).\u0026nbsp;In addition to soil\u0026nbsp;available C and N, a number of studies are available on soil N\u003csub\u003e2\u003c/sub\u003eO emission in response to soil variables, microbial community composition, and key functional genes controlling\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO production and consumption (Qin et al., 2018; Kong et al., 2021; Wei et al., 2021; Lin et al., 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmmonium oxidation controlled by ammonia-oxidizing archaea and bacteria (AOA-\u003cem\u003eamoA\u003c/em\u003e and AOB-\u003cem\u003eamoA\u003c/em\u003e) is one of the main limiting steps of nitrification (Tokutomi et al., 2010; Zou et al., 2022). Moreover, the conversion of nitrite into nitric oxides by \u003cem\u003enirS\u003c/em\u003e, \u003cem\u003enirK\u0026nbsp;\u003c/em\u003egenes and N\u003csub\u003e2\u003c/sub\u003eO into dinitrogen by \u003cem\u003enosZ\u003c/em\u003e gene are the main limiting steps\u0026nbsp;of denitrification (Zhou et al., 2020; Li et al., 2021). Several studies investigated the responses of these genes and N\u003csub\u003e2\u003c/sub\u003eO emissions to N addition and flooded conditions (Shaaban et al., 2018; Qin et al., 2018; Zhou et al., 2020; Yang et al., 2022). Scholars also emphasized the importance of these key factors when investigating soil N\u003csub\u003e2\u003c/sub\u003eO emissions.\u0026nbsp;Future works should determine whether rice cultivation influences the production and consumption of N\u003csub\u003e2\u003c/sub\u003eO and the abundance of several important functional genes, which could affect soil N\u003csub\u003e2\u003c/sub\u003eO release (Ma et al., 2008; Yang et al., 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRice-based cultivation in China is critical in regulating soil N\u003csub\u003e2\u003c/sub\u003eO emission, and the effect of rice plantation on several key functional genes that regulate N\u003csub\u003e2\u003c/sub\u003eO emission has not been extensively studied across different rice cultivation modes. In this regard, the present study selected two frequently rice cultivation rotation systems, namely, upland rice\u0026ndash;rapeseed (UR) and flooded rice\u0026ndash;rapeseed (RR), to investigate the effect of rice plantations on soil N\u003csub\u003e2\u003c/sub\u003eO release (during the rice-growing seasons). This study aimed to i) assess the contribution of soil available C and N fractions related to N\u003csub\u003e2\u003c/sub\u003eO emissions (i.e., soil DOC, mineral N, MBC and MBN contents) in two rice-based rotation systems and ii) explore the potential relationship between N\u003csub\u003e2\u003c/sub\u003eO emissions and soil-related C and N fractions and microbial functional genes. We hypothesized that rice plantation would change soil available C and N fractions due to their absorption and utilization of active C secretion, consequently affecting the abundance of key functional genes related to N\u003csub\u003e2\u003c/sub\u003eO production and consumption regardless of rice-based rotations. Thus, several key environmental factors, soil characteristics, and microbial functional genes corresponding to soil N\u003csub\u003e2\u003c/sub\u003eO production and consumption were investigated.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDescription of study site and design of field experiments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental field in HuangGang Academy of Agricultural Sciences, Hubei Province, China (30\u0026deg;63\u0026apos;N, 114\u0026deg;87\u0026apos;E), was selective to carry out the field trial. Area where the trial site is located has a subtropical monsoon environment with mean yearly temperature and precipitation of 18 \u0026deg;C and 1100 mm, respectively. Table S1 shows the soil physicochemical properties including texture, bulk density (BD) total organic C, total N, and pH at the onset of the study.\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted from May 2019 to September 2020, which included two rice plantation seasons (from May 2019 to September 2019 and from May 2020 to September 2020, respectively) and one rapeseed season (from October 2019 to May 2020). Real data on N\u003csub\u003e2\u003c/sub\u003eO emission from January to March 2020 during the rapeseed season were missing due to the epidemic of COVID-19. Two rice cultivation rotation modes, namely, UR and RR, were selected.\u0026nbsp;For the UR model, which means rapeseed\u0026ndash;aerobic rice rotation, aerobic rice was cultivated in summer without flooding, while for RR model, which represents rapeseed\u0026ndash;flooded rice rotation, paddy rice seedlings were transplanted in late May. For both UR and RR rotations, winter rapeseed seeds were sown in early October.\u003c/p\u003e\n\u003cp\u003eA split-plot experiment was conducted in a plot area of 24 m\u003csup\u003e2\u003c/sup\u003e (4 m \u0026times; 6 m). Within each plot, a 1.5 m \u0026times;1.5 m area was implemented with conventional fertilization but without rice plantation (bare soil with conventional fertilization) to confirm the effect of rice plantation and soil conditions, a part of the non-rice treatments during the rice season. Each treatment was repeated three times. Four treatments were used in total: 1) conventional upland rice\u0026ndash;rapeseed rotation (UR-CC); 2) upland rice\u0026ndash;rapeseed without rice plantation (UR-NC); (3) conventional flooded rice\u0026ndash;rapeseed (RR-CC); and (4) flooded rice\u0026ndash;rapeseed without rice plantation (RR-NC).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe fertilizer applied during the upland rice season and the flooded rice season consisted of 225 kg N ha\u003csup\u003e-1\u003c/sup\u003e, 112 kg P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u0026nbsp;\u003c/sub\u003eha\u003csup\u003e-1\u003c/sup\u003e, and 225 kg K\u003csub\u003e2\u003c/sub\u003eO ha\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;All K and P fertilizer dosages were applied as base fertilizer along with one-half of the N fertilizer dose, and the other half was applied as topdressing. During the rapeseed season, the fertilizer applied included 192 kg N ha\u003csup\u003e-1\u003c/sup\u003e, 112 kg P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u0026nbsp;\u003c/sub\u003eha\u003csup\u003e-1\u003c/sup\u003e, and 112 kg K\u003csub\u003e2\u003c/sub\u003eO ha\u003csup\u003e-1\u003c/sup\u003e. All K and P fertilizers were applied as base fertilizer, and only one component of the N fertilizer (60%) was applied as base fertilizer. The leftover portion of N was applied as topdressing. Table S2 shows detailed information on fertilizer application and management practices for the treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eN\u003csub\u003e2\u003c/sub\u003eO flux measurement\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO gas samples were collected from the treated areas by using static opaque chamber technique (Wang et al., 2013). The chamber was made up of a foundation frame and a chamber lid.\u0026nbsp;The chamber cover, which is covered in insulating foam, has a bottom area of 0.25 m\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eand a height of 1 m, while the base structure has the same bottom area as the chamber and a height of 0.3 m. For gas sampling during the rapeseed growth season, another chamber with the height of 0.5 m was combined with the chamber with the height of 1 m when the rapeseed grew up more than 1 m. After plowing the plot at the start of each growing season, the foundation frame was inserted manually into the soil. An upper edge that can be filled with water was specifically designed for each base to maintain the chamber\u0026rsquo;s liquid closure prior to gas sampling. Five tubes of gas samples were manually taken from each chamber by using 50 mL syringes, and a 10 min gap was implemented between each sampling event.\u003c/p\u003e\n\u003cp\u003eGas was collected twice weekly, but the frequency was adjusted to seven times a week following fertilization and heavy rain. Within 24 hours of collection, gas sample concentrations were determined using a gas chromatography (GC; 7890A, Agilent Technologies, California, USA) system fitted with an electron capture detector (ECD). Following the measurement, N\u003csub\u003e2\u003c/sub\u003eO fluxes and seasonal total N\u003csub\u003e2\u003c/sub\u003eO emissions from each plot were calculated\u0026nbsp;using the approaches proposed by Zheng et al. (2008). The data of N\u003csub\u003e2\u003c/sub\u003eO emission fluxes during the first rice-cultivated season in 2019 for RR-CC treatment were obtained from the study of Xu et al. (2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuxiliary parameter measurements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOther key variables were determined (Xu et al., 2022a). A small digital thermometer was used to capture ambient temperature inside each compartment (JM624, Liwen Electronics Ltd., Tianjin, China). A vertical ruler was used to measure the field inundation water level (only for the rice-planting season in the RR system). Other crucial factors were measured concurrently with the monitoring of N\u003csub\u003e2\u003c/sub\u003eO flow (Xu et al., 2022a). During gas sample collection, the temperature of the soil at a 5\u0026nbsp;cm depth was manually measured using an electronic probe. Each week, five random topsoil (0-20 cm) samples were collected from each plot and manually blended to produce a single composited soil sample. The sample was quickly transported to the laboratory and stored in a refrigerator at 4 ℃. A portion of soil samples was used to measure soil water content. Another part of soil samples was used to determine soil pH, available C and N contents suggested by Xu et al. (2022b). Another part of the soil samples was used to determine soil microbial biomass C and N (MBC and MBN) by chloroform fumigation method, which was proposed and modified by Brookes et al. (1987). Soil bulk density (BD) was determined using columnar soil samples collected using cylinder rings at the beginning of the experiment. A modest auto-monitoring meteorological station located close to the experimental location was used to record air temperature and precipitation daily.\u0026nbsp;Water filled pore space (WFPS), which describes the soil moisture during the crop-growing period for the UR system, was computed by the following equation:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere 2.65 g cm\u003csup\u003e\u0026minus;3\u0026nbsp;\u003c/sup\u003ewas assumed as soil particle density (PD). The partial data of soil parameters during the first rice-planting season in 2019 for RR-CC treatment were obtained from the study of Xu et al. (2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eReal-time quantitative PCR (qPCR)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe abundance of several key functional genes involved in N\u003csub\u003e2\u003c/sub\u003eO production and consumption (\u003cem\u003eamoA\u003c/em\u003e,\u003cem\u003e\u0026nbsp;nirS\u003c/em\u003e, \u003cem\u003enirK\u003c/em\u003e, and \u003cem\u003enosZ\u003c/em\u003e genes) was quantitatively determined by real-time quantitative PCR procedure (qPCR) using the referenced primer sets (Table S3). Approximately 0.35 g of fresh soil was sifted through a 2 mm sieve and used to extract soil microbial DNA with Fast DNA SPIN Kit (MP Biomedicals, USA) based on the manufacturer\u0026rsquo;s instructions. After microbial DNA extraction, qPCR analysis was performed by SYBR Green method. The measurement range and the \u003cem\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e value of each calibration curve were set at 95% to 110% and higher than 0.98, respectively. The total volume of 15 \u0026micro;L of the quantitative PCR reaction liquid was composed of 7.5 \u0026micro;L of 2\u0026times;SYBR Green Mix (TaKaRA Japan), 1 \u0026micro;L of diluted template DNA, and 0.7 \u0026micro;L of primer, and the remaining volume was sterile water. The amplification conditions described in a previous study (Xu et al., 2022b) was referenced to DNA amplification. The formulae mentioned by Macdonald et al. (2011) were used to calculate the copy number of each gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData displaying normality were used directly, while data that had non-normality were analyzed and log-transformed before use.\u0026nbsp;Cumulative seasonal N\u003csub\u003e2\u003c/sub\u003eO emissions were compared among the four treatments by using the analysis of variance (ANOVA) procedure. Functional relationships among N\u003csub\u003e2\u003c/sub\u003eO emission fluxes and soil parameters, and N\u003csub\u003e2\u003c/sub\u003eO production and consumption-related functional genes were described using linear or nonlinear regression. Structural equation models (SEM) were used to exhibit the direct and indirect effects of aforementioned factors on N\u003csub\u003e2\u003c/sub\u003eO emissions by using AMOS software (AMOS 21.0, SPSS Inc., Chicago, USA). Additionally, SPSS (version 19.0, SPSS Inc., Chicago, USA) and Origin 8.0 tools (Origin Lab Corporation, USA) were used for figure creation and statistical analysis. Finally, a \u003cem\u003ep \u0026le; 0.05\u003c/em\u003e level was set to indicate a significantly statistical difference.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClimate, environmental, and soil variables\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;The daily air and soil temperatures at 5 cm depth ranged from -3.3 \u0026deg;C to 33.6 \u0026deg;C and from 4.8 \u0026deg;C to 32.7 \u0026deg;C, respectively, over the experimental period. The recorded annual precipitation levels were 1103.9 and 559.3 mm during the 2019\u0026ndash;2020 period and the following rice season in 2020, respectively (Fig. S1). During the first and following upland rice-planting seasons, soil WFPS varied from 35.6 to 68.8% and 37.4 to 64.7% in the UR-CC treatment and 34.1 to 69.2% and 36.7 to 62.9% in the UR-NC treatment, respectively (Fig. S1). During the first and following flooded rice-planting seasons, the flood water depths were 0\u0026ndash;4.65 and 0\u0026ndash;4.72 cm in the RR-CC treatment and 0\u0026ndash;4.55 and 0\u0026ndash;4.62 cm in the RR-NC treatment, respectively (Fig. S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the initial and succeeding upland rice cultivation seasons, the soil DOC ranged from 20 mg C kg\u003csup\u003e-1\u003c/sup\u003e to 50 mg C kg\u003csup\u003e-1\u003c/sup\u003e for the UR-CC and UR-NC treatments. For the RR-CC and RR-NC treatments, the soil DOC ranged from 20 mg C kg\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eto 60 mg C kg\u003csup\u003e-1\u003c/sup\u003e during both flooded rice-planting seasons (Fig. 1a). The average soil DOC content in the UR rotation system was comparable with that in the RR rotation system (Table S4). For all treatments, the soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content increased sharply with basal N fertilization and subsequently decreased to a low level. The lowest soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content\u0026nbsp;was\u0026nbsp;approximately 4 mg N kg\u003csup\u003e-1\u003c/sup\u003e, while the highest value was more than\u0026nbsp;90 mg N kg\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eduring the upland rice cultivation season in UR rotation. The corresponding lowest value was\u0026nbsp;approximately 1 mg N kg\u003csup\u003e-1\u003c/sup\u003e, and the highest value was more than 65 mg N kg\u003csup\u003e-1\u003c/sup\u003e during the flooded rice cultivation seasons in RR rotation. The average soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content was higher in the UR rotation system than in the RR rotation system (Fig. 1b). The soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N content increased with basal N fertilization and subsequently decreased to a low level (Fig. 1c). The average soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N contents were significantly higher in UR rotation than in RR rotation (Table S4).\u003c/p\u003e\n\u003cp\u003eFor all treatments, soil MBC content dynamics exhibited similar patterns for the two upland rice growing seasons as well as for the two flooded rice growing seasons. The highest average soil MBC content was detected in the UR-CC treatment, with ranges of 74.91\u0026ndash;638.20 and 160.38\u0026ndash; 625.07 mg C kg\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eduring the first and following upland rice cultivation seasons, respectively (Fig. 2a). Meanwhile, the highest average soil MBN content was observed in the UR-CC treatment, with ranges of 20.21\u0026ndash;46.40 and 13.42\u0026ndash;45.35 mg N kg\u003csup\u003e-1\u003c/sup\u003e during the first and following upland rice cultivation seasons, respectively (Fig. 2b). The MBC/MBN ratio was higher in RR rotation than in UR rotation (Fig. 2c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eN\u003csub\u003e2\u003c/sub\u003eO emissions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ein the UR-CC treatment, the N\u003csub\u003e2\u003c/sub\u003eO emission fluxes were -8.89\u0026ndash;644.59 and 1.92\u0026ndash;1536.22 \u0026mu;g N m\u003csup\u003e-2\u003c/sup\u003e h\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eduring the first and following upland rice-planting seasons, respectively. Higher N\u003csub\u003e2\u003c/sub\u003eO emission fluxes of -1.13\u0026ndash;1196.43 and -19.32\u0026ndash;1607.49 \u0026mu;g N m\u003csup\u003e-2\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e were observed in the UR-NC treatment compared with those in the UR-CC treatment. In RR rotation, the N\u003csub\u003e2\u003c/sub\u003eO emission fluxes were -4.06\u0026ndash;564.65 and -18.12\u0026ndash;803.22 \u0026mu;g N m\u003csup\u003e-2\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e in the RR-CC treatment during the flooded rice-planting seasons. Higher N\u003csub\u003e2\u003c/sub\u003eO emission fluxes were observed in the RR-NC treatment compared with those in the RR-CC treatment, with ranges of 3.68\u0026ndash;664.65 and 3.65\u0026ndash;721.02 \u0026mu;g N m\u003csup\u003e-2\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e, respectively (Fig. 3).\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;seasonal cumulative\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO emissions\u0026nbsp;in the UR-CC treatment were 1.54 \u0026plusmn; 0.16 and 2.57 \u0026plusmn; 0.28 kg N ha\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003efor the first and second upland rice-planting seasons, respectively, which were significantly lower than those in the UR-NC treatment, which had total N\u003csub\u003e2\u003c/sub\u003eO emissions of 2.45 \u0026plusmn; 0.07 and 3.74 \u0026plusmn; 0.37 kg N ha\u003csup\u003e-1\u003c/sup\u003e, respectively (embedded graph in Fig. 3). However, compared with UR rotation, RR rotation had lower\u0026nbsp;seasonal cumulative\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO emissions of 0.71 \u0026plusmn; 0.20 and 0.76 \u0026plusmn; 0.04 kg N ha\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ein the RR-CC treatment and 1.43 \u0026plusmn; 0.35 and 1.16 \u0026plusmn; 0.08 kg N ha\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ein the RR-NC treatment during the first and following flooded rice-planting seasons, respectively (embedded graph in Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAbundance of AOA-amoA, AOB-amoA, nirK,\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003enirS, and nosZ genes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor UR and RR rotations, the abundance of the AOB-\u003cem\u003eamoA\u0026nbsp;\u003c/em\u003egene was approximately two orders of magnitude lower than that of the AOA-\u003cem\u003eamoA\u0026nbsp;\u003c/em\u003egene regardless of the rice planting season (Fig. 4). The average copy number of the AOA-\u003cem\u003eamoA\u0026nbsp;\u003c/em\u003egene during the upland rice or flooded rice cultivation seasons for the rice planting treatments was significantly lower than those for the non-rice planting treatments (Fig. 4a).\u0026nbsp;The average copy number of the AOB-\u003cem\u003eamoA\u0026nbsp;\u003c/em\u003egene during the upland rice cultivation seasons for the UR-CC treatment was significantly lower than that for the UR-NC treatment (Fig. 4b). The average copy number of the AOB-\u003cem\u003eamoA\u0026nbsp;\u003c/em\u003egene in the RR-CC treatment during the flooded rice-growing seasons was higher than that in the RR-NC treatment (Table S5). In particular, the gene copy number ratio of the AOA-\u003cem\u003eamoA\u003c/em\u003e to AOB-\u003cem\u003eamoA\u003c/em\u003e (AOA/AOB) was significantly higher in UR rotation than in RR rotation (Fig. 4c, Table S5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe average \u003cem\u003enirK\u003c/em\u003egene copy number in UR rotation during the upland rice-growing seasons was significantly lower than those in RR rotation during both flooded rice-growing seasons (Fig. 4e, Table S6). The average copy number of the \u003cem\u003enirS\u003c/em\u003egenes in the RR-CC treatmentduring the flooded rice-growing seasons were significantly lower than that in the RR-NC treatment (Fig. 4d, Table S6). For upland rice-growing seasons, the average copy number of the \u003cem\u003enirS\u003c/em\u003e\u003cem\u003e\u0026nbsp;and nirK\u003c/em\u003e genes was comparable between the rice-planted and non-rice treatments under UR rotation (Fig. 4d, e, Table S6). For RR rotation, the average copy number of the \u003cem\u003enosZ\u003c/em\u003egene was higher in rice-planted treatments than in non-rice-planted treatments (Fig. 4f, Table S6). In UR and RR rotations, the value of \u003cem\u003enosZ\u003c/em\u003e/(\u003cem\u003enirS\u003c/em\u003e+\u003cem\u003enirK\u003c/em\u003e) was higher in rice-planted treatments than in non-rice-planted treatments (Fig. 4g, Table S6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRelationship between\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eN\u003csub\u003e2\u003c/sub\u003eOemissions and\u0026nbsp;soil parameters and\u0026nbsp;functional\u0026nbsp;genes\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Fig. 5, the soil N\u003csub\u003e2\u003c/sub\u003eO flux increased linearly with increasing soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and DOC concentrations in the RR-CC and RR-NC\u0026nbsp;treatments. The N\u003csub\u003e2\u003c/sub\u003eO fluxes in soils treated with UR-CC and UR-NC\u0026nbsp;exponentially increased with increasing\u0026nbsp;soil DOCcontent (Fig. 5a) and linearly\u0026nbsp;increased with increasing soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e level (Fig. 5b). Logarithmic relationships were observed between N\u003csub\u003e2\u003c/sub\u003eO fluxes and the value of DOC/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e for various treatments during the rice-growing seasons (Fig. 5c). Furthermore,\u0026nbsp;positive linear relationships between\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO\u0026nbsp;fluxes and the value of AOA /AOB were observed\u0026nbsp;in UR rotation\u0026nbsp;(Fig. 5d). In RR rotation,\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO\u0026nbsp;fluxes were positively linearly related to the copy number of the \u003cem\u003enirS\u003c/em\u003e gene (Fig. 5e) but negatively linearly related to the copy number of the \u003cem\u003enosZ\u003c/em\u003e gene (Fig. 5f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSEM describing the effect of key factors on N\u003csub\u003e2\u003c/sub\u003eO emissions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 6, an SEM was applied to describe the direct and indirect effects of several key components on soil N\u003csub\u003e2\u003c/sub\u003eO emissions. In particular, soil moisture and/or soil temperature, soil C and N availability, and functional genes (AOA-\u003cem\u003eamoA,\u0026nbsp;\u003c/em\u003eAOB-\u003cem\u003eamoA\u003c/em\u003e,and their ratio) explained 76% and 81% of soil N\u003csub\u003e2\u003c/sub\u003eO emissions in the UR-CC and UR-NC treatments, respectively (Fig. 6a and b). Meanwhile, soil temperature and soil moisture, soil C and N availability, and functional genes (\u003cem\u003enirS\u003c/em\u003e and \u003cem\u003enosZ\u003c/em\u003e) explained 79% and 83% of soil N\u003csub\u003e2\u003c/sub\u003eO emissions in the RR-CC and RR-NC treatments, respectively (Fig. 6c and d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eYields from different crop seasons\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S7 shows that the rapeseed yield had no significant difference between the UR-CC and RR-CC treatments throughout the rapeseed-growing season, with yields amounting to 5.67 \u0026plusmn; 0.66 and 5.95 \u0026plusmn; 0.60 t ha\u003csup\u003e-1\u003c/sup\u003e, respectively. The rice yields in the UR-CC treatment during the first and following rice-growing seasons were 7.36 \u0026plusmn; 0.48 and 7.58 \u0026plusmn; 0.29 t ha\u003csup\u003e-1\u003c/sup\u003e, respectively, which were higher than those in the RR-CC treatment (6.80 \u0026plusmn; 0.54 and 6.72 \u0026plusmn; 0.69 t ha\u003csup\u003e-1\u003c/sup\u003e, respectively).\u0026nbsp;Furthermore, higher response of N\u003csub\u003e2\u003c/sub\u003eO emissions to per unit rice yield was observed in the UR-CC treatment than that in the RR-CC treatment for both rice-growing seasons (0.21 \u0026plusmn; 0.01 \u003cem\u003evs\u003c/em\u003e.0.10 \u0026plusmn; 0.02 and 0.34 \u0026plusmn; 0.03 \u003cem\u003evs\u003c/em\u003e. 0.11 \u0026plusmn; 0.01, respectively).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEffects of rice-based rotation on soil N\u003csub\u003e2\u003c/sub\u003eO emissions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent cropping patterns have a significant effect on N\u003csub\u003e2\u003c/sub\u003eO emissions from rice fields (Zhou et al., 2017; Zhou et al., 2022b; Cheng et al., 2022; Xu et al., 2023).\u0026nbsp;Regardless of rice planting, N\u003csub\u003e2\u003c/sub\u003eO emissions from the tested soils were higher during the upland rice-growing seasons than during the flooded rice-growing seasons. A field trial revealed significantly higher seasonal N\u003csub\u003e2\u003c/sub\u003eO emissions from cultivated aerobic rice soil than that from soils planted with flooded rice (Mohanty et al., 2017). These differences could be attributed to the large variations in field water management between upland rice and flooded rice cultivation, which further influenced soil N\u003csub\u003e2\u003c/sub\u003eO emissions (Liu et al., 2010; Shaaban et al., 2018). In the present study, the moisture content (WFPS) of upland paddy soil was 45%\u0026ndash;60%, which is conducive to nitrification by soil microorganisms, thereby promoting soil N\u003csub\u003e2\u003c/sub\u003eO emission (Qin et al., 2018). The highest soil N\u003csub\u003e2\u003c/sub\u003eO emission occurred when the soil WFPS ranged from 45% to 75%, where nitrifying microorganisms had vigorous activities (S\u0026aacute;nchez-Mart\u0026iacute;n et al., 2008). In the UR system, soil N\u003csub\u003e2\u003c/sub\u003eO emission fluxes during the rice season were related to the AOA/AOB ratio in the UR-CC and UR-NC treatments, indicating that soil N\u003csub\u003e2\u003c/sub\u003eO emissions were mainly contributed by nitrification dominated by AOA. Wang et al. (2021) reported that although organic material amendments were added to upland soil, N\u003csub\u003e2\u003c/sub\u003eO loss derived from denitrification was negligible. In addition, the depth of the water layer during the rice-growing season is an important factor affecting soil N\u003csub\u003e2\u003c/sub\u003eO emissions (Xu et al., 2022a, b). Regardless of drainage during the mid-season and after maturity, the paddy field typically remained flooded during the rice season in RR mode, which was conducive to the progress of denitrification (Di et al., 2014). Prolonged flooding promoted complete denitrification, indicating the conversion of N\u003csub\u003e2\u003c/sub\u003eO into N\u003csub\u003e2\u003c/sub\u003e. This finding was confirmed by the relationship between N\u003csub\u003e2\u003c/sub\u003eO and denitrifying functional gene \u003cem\u003enosZ\u003c/em\u003e (Fig. 5f). In contrast, higher inorganic N levels were observed in UR mode than in RR mode, which may have provided a sufficient substrate for nitrifying and denitrifying microbial activities. Our study revealed that inorganic N affected functional genes related to nitrification, thereby affecting N\u003csub\u003e2\u003c/sub\u003eO production (Fig. 6). Moreover, the average soil DOC content during the rice season was higher in RR rotation than in UR rotation, regardless of rice plantation. Previous investigations suggested soil DOC as an active substrate that can be utilized by microorganisms effectively and thus influenced N\u003csub\u003e2\u003c/sub\u003eO production and emissions (Sanchez-Martin et al., 2008; Shaaban et al., 2019). For example, a higher DOC content can promote soil denitrification to regulate N\u003csub\u003e2\u003c/sub\u003eO emissions (Lee et al., 2017; Zhou et al., 2017b). Apart from the RR-NC treatment, N\u003csub\u003e2\u003c/sub\u003eO emission rates during the rice-growing seasons had a positive association with the value of soil DOC/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (Fig. 5c), consistent with the results of previous studies (Hu et al., 2015; Zhou et al., 2017b). Further, Lan et al. (2017) discovered that the value of DOC/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e should be between 0.69 and 0.84 for the N\u003csub\u003e2\u003c/sub\u003eO emission peak to occur. Zhou et al. (2017b) reported that N\u003csub\u003e2\u003c/sub\u003eO fluxes had a curvilinear relationship with soil DOC/(NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e+NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e). These results illustrate that the ratio of soil labile C to mineral N can regulate N\u003csub\u003e2\u003c/sub\u003eO emissions from paddy fields.\u003c/p\u003e\n\u003cp\u003eThe differences in physiology between upland rice and flooded rice soils could be attributed to their different cropping patterns (sowing \u003cem\u003evs\u003c/em\u003e. transplantation). For instance, compared with flooded rice, upland rice had a higher planting density but considerably smaller roots and stems. Moreover, upland rice had a higher grain yield than flooded rice (Table S7). These differences may further contribute to different nutrient utilization patterns in the studied soils during the two rice-growing seasons, thereby affecting soil C and N cycling and N\u003csub\u003e2\u003c/sub\u003eO production and consumption (Mohanty et al., 2017; Zhang et al., 2022; Xu et al., 2023). In the RR-CC treatment, upland rice with finer roots and higher planting density and grain yield required more mineral N, which might have competed with N\u003csub\u003e2\u003c/sub\u003eO production activities for the common N substrate (Kim et al., 2021). However, in the UR-CC treatment, enhanced N mineralization during upland rice planting period might be enhanced by the following pathways: (1) higher soil DIN and MBN used as substrates by N\u003csub\u003e2\u003c/sub\u003eO production-related microbes and (2) optimum soil aeration conditions in comparison with that during the flooded rice-plantation period. Compared with flooded padded soils, upland rice may have higher grain yield and substantial organic N mineralization, which contribute to greater N\u003csub\u003e2\u003c/sub\u003eO emissions.\u003c/p\u003e\n\u003cp\u003eIn UR rotation, higher AOA\u003cem\u003e-amoA\u0026nbsp;\u003c/em\u003egene abundance was found, however, N\u003csub\u003e2\u003c/sub\u003eO flux was not significantly related to the abundance of the AOA-\u003cem\u003eamoA\u003c/em\u003e gene but was highly correlated with that of the AOB\u003cem\u003e-amoA\u003c/em\u003e gene (Fig. 6) and/or AOA/AOB (Fig. 5d and 6). Moreover, AOB-\u003cem\u003eamoA\u003c/em\u003e gene abundance and AOA/AOB were indirectly correlated with N\u003csub\u003e2\u003c/sub\u003eO flux by the middle bridge of soil mineral N contents. In particular, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N was strongly and positively correlated with the AOB-\u003cem\u003eamoA\u003c/em\u003e gene, which was associated with N\u003csub\u003e2\u003c/sub\u003eO emission (Fig. 6a). Our results indicate that AOB may play a great role in the microbial release of N\u003csub\u003e2\u003c/sub\u003eO during upland rice growing seasons with lower soil moisture, consistent with the previous findings that AOB may be the primary\u0026nbsp;producers of N\u003csub\u003e2\u003c/sub\u003eO in alkaline pH soils (Zou et al. 2022). Pan et al. (2016) found that AOB plays a dominant role in the microbial release of N\u003csub\u003e2\u003c/sub\u003eO in grassland soils.\u003c/p\u003e\n\u003cp\u003eIn RR rotation, the N\u003csub\u003e2\u003c/sub\u003eO emission flux showed a positive correlation with the denitrifying functional gene\u0026nbsp;\u003cem\u003enirS\u003c/em\u003e, consistent with previous experiments (Wang et al., 2022).\u0026nbsp;The \u003cem\u003enirS\u003c/em\u003e gene has a stronger effect on N\u003csub\u003e2\u003c/sub\u003eO production and emission in soil than the \u003cem\u003enirK\u003c/em\u003e gene (Qin et al., 2020; Zhou et al., 2020). However, other works reported that the higher abundance of the \u003cem\u003enirK\u003c/em\u003e gene in soil under flooded rice growing periods may contribute to greater N\u003csub\u003e2\u003c/sub\u003eO emissions. Yang et al. (2022) found the high abundance of the \u003cem\u003enirK\u003c/em\u003e gene and the accompanied greater N\u003csub\u003e2\u003c/sub\u003eO emissions. Basing on these results, we suggest that the role of \u003cem\u003enirS\u003c/em\u003e and \u003cem\u003enirK\u003c/em\u003e genes in N\u003csub\u003e2\u003c/sub\u003eO emissions needs to be further explored in paddy fields in future research. According to Chen et al. (2020), the (\u003cem\u003enirS\u003c/em\u003e+\u003cem\u003enirK\u003c/em\u003e)/\u003cem\u003enosZ\u003c/em\u003e value denotes the genetic capacity of denitrifying genes that controls whether N\u003csub\u003e2\u003c/sub\u003eO is converted into N\u003csub\u003e2\u003c/sub\u003e or released into the environment as a greenhouse gas. In UR and RR rotation systems, the value of (\u003cem\u003enirS\u003c/em\u003e+\u003cem\u003enirK\u003c/em\u003e)/\u003cem\u003enosZ\u0026nbsp;\u003c/em\u003ewas higher in non-rice-planted treatments than in rice-planted treatments, which may further explain the greater N\u003csub\u003e2\u003c/sub\u003eO emissions in the former. In agreement with our results, previous studies attributed the higher N\u003csub\u003e2\u003c/sub\u003eO emissions to higher (\u003cem\u003enirS\u003c/em\u003e+\u003cem\u003enirK\u003c/em\u003e)/\u003cem\u003enosZ\u003c/em\u003e (Kong et al., 2021; You et al. 2022). Thus,\u0026nbsp;the ratio of (\u003cem\u003enirS\u003c/em\u003e+\u003cem\u003enirK\u003c/em\u003e)/\u003cem\u003enosZ\u0026nbsp;\u003c/em\u003eshould be considered when investigating soil N\u003csub\u003e2\u003c/sub\u003eO emission in rice-based field systems in the future.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEffects of rice planting on soil N\u003csub\u003e2\u003c/sub\u003eO emission\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, N\u003csub\u003e2\u003c/sub\u003eO emissions in UR and RR rotation systems were significantly greater in the non-rice planting treatment than in the rice planting treatment, consistent with previous findings (Kim et al., 2021). The average seasonal N\u003csub\u003e2\u003c/sub\u003eO emission rate from paddy soil with plants was lower than that without plants (L\u0026oacute;pez-Fern\u0026aacute;ndez et al., 2007) because of lower denitrification potential (Kim et al., 2021). Rice growth was presumed to compete for N substrate, which further supported nitrifying and denitrifying microorganisms after rice transplanting. Therefore, non-rice-planted soil with high N might provide sufficient substrates for N\u003csub\u003e2\u003c/sub\u003eO production in microorganisms and enhanced N\u003csub\u003e2\u003c/sub\u003eO emissions from the soil (Opez-Fernande, 2007; Davidson, 2009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe average soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N concentration was significantly higher in the non-rice treatment than in the crop-planting treatment, providing active substrate and supporting the processes related to N\u003csub\u003e2\u003c/sub\u003eO production. In agreement with our findings, Hodge et al. (2000) discovered that inorganic N concentration was higher in bare soil than in soil cultivated with crops, and it offered a sufficient substrate for nitrifying and denitrifying microorganisms and promoted the synthesis and emission of N\u003csub\u003e2\u003c/sub\u003eO from the soil (Xing et al., 1998; Opez-Fernande, 2007; Zhang et al., 2016). The average MBC and MBN contents in the soil under cropping were higher than those in non-cropping soil, indicating that rice cultivation increased microbial activity, which may promote N\u003csub\u003e2\u003c/sub\u003eO production to some extent. Exudation from rice roots as an organic C sources (Zhu et al., 2018, Khan et al., 2019) provide remarkable energy for microbial denitrification and facilitate N\u003csub\u003e2\u003c/sub\u003eO reduction. However, the negative effect of rice plantation on N\u003csub\u003e2\u003c/sub\u003eO emissions could be expected due to O\u003csub\u003e2\u003c/sub\u003e consumption and favored denitrification during flooded rice-growing period, leading to the reduction of N\u003csub\u003e2\u003c/sub\u003eO into N\u003csub\u003e2\u003c/sub\u003e (Yang et al., 2021). In a double rice-fallow cultivation mode, Xu et al. (2022b) observed that N\u003csub\u003e2\u003c/sub\u003eO emissions were not different between rice-plantation and non-rice plantation treatments.\u003c/p\u003e\n\u003cp\u003eRice cropping affects functional genes related to nitrification and denitrification, thereby affecting N\u003csub\u003e2\u003c/sub\u003eO emissions (Ma et al., 2008; Yang et al., 2017). The average copy number of the AOA-\u003cem\u003eamoA\u003c/em\u003e gene in soil was higher in the non-rice treatments than in the rice plantation treatments\u0026nbsp;in the UR and RR rotation systems. The copy number of the AOA-\u003cem\u003eamoA\u003c/em\u003e gene was higher by two orders of magnitude than that of the AOB-\u003cem\u003eamoA\u003c/em\u003e gene in UR and RR rotations, highlighting the ecological niche of AOA that occupied the studied soils. Previous studies reported that AOA dominated the paddy soils compared with AOB (Chen et al., 2008).\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003enosZ\u003c/em\u003e gene in soil in the RR-CC treatment was higher than that in the RR-NC treatment. The higher abundance of the \u003cem\u003enosZ\u003c/em\u003e gene may lead to higher nitrous oxide reductase, which modulates the reduction of N\u003csub\u003e2\u003c/sub\u003eO into N\u003csub\u003e2\u003c/sub\u003e, consequently reducing N\u003csub\u003e2\u003c/sub\u003eO emissions (Liu et al., 2010; Xu et al., 2022b). As proposed previously, the prevalence of\u0026nbsp;\u003cem\u003enosZ\u003c/em\u003e-containing microorganisms may be related to the ambient N\u003csub\u003e2\u003c/sub\u003eO concentration because N\u003csub\u003e2\u003c/sub\u003eO serves as the electron receptor for nitrous oxide reductase (Qin et al., 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRice plantations enhanced N\u003csub\u003e2\u003c/sub\u003eO emissions mainly by increasing the abundance of the AOB\u003cem\u003e-amoA\u0026nbsp;\u003c/em\u003eand/or the value of AOA/AOB in a typical RR rotation system. The increase in N\u003csub\u003e2\u003c/sub\u003eO emissions could be due to the increasing abundance of the\u0026nbsp;\u003cem\u003enirS\u003c/em\u003e gene and the reducing abundanceofthe\u0026nbsp;\u003cem\u003enosZ\u003c/em\u003e gene in a typical flooded RR rotation system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImplications for\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003efuture studies\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRice cultivation has a significant effect on soil N\u003csub\u003e2\u003c/sub\u003eO emissions. In theory, our findings showed that functional genes related to nitrification and denitrification interacted with important soil factors to govern N\u003csub\u003e2\u003c/sub\u003eO emission.\u0026nbsp;However, soil N\u003csub\u003e2\u003c/sub\u003eO production and consumption may co-occur in rice-based fields\u0026nbsp;that are characterized by changing redox conditions, substrates, and microbial\u0026nbsp;conditions during the rice-growing period. We propose that certain significant environmental variables, soil characteristics, and microbes regulating N\u003csub\u003e2\u003c/sub\u003eO production and consumption will work together to control field-level release of soil N\u003csub\u003e2\u003c/sub\u003eO.\u0026nbsp;As a result, studies should focus on variations in available C and N substrates as well as microorganisms associated with N\u003csub\u003e2\u003c/sub\u003eO generation, which could be affected by the changing soil condition and habitat in a rice-based field. In summary, rice planting could be beneficial to the mitigation of soil N\u003csub\u003e2\u003c/sub\u003eO emissions compared with soil without rice planted but spread with N fertilizer unintentionally.\u0026nbsp;Future\u0026nbsp;studies should investigate soil N\u003csub\u003e2\u003c/sub\u003eO emissions in paddy fields in terms of\u0026nbsp;differences\u0026nbsp;in\u0026nbsp;the\u0026nbsp;characteristics of N\u003csub\u003e2\u003c/sub\u003eO production-related microorganisms and\u0026nbsp;functional genes.\u0026nbsp;For instance, the microbial effect on\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO emissions\u0026nbsp;could be further investigated through RNA-based microbial profiling (Wei et al. 2021). Moreover, \u003csup\u003e15\u003c/sup\u003eN-\u003csup\u003e18\u003c/sup\u003eO labeling approach can be used to resolve the quantity of microbial denitrification processes related to\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO production, similar to conventional denitrification and nitrifier denitrification (Kool et al. 2010).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study examined N\u003csub\u003e2\u003c/sub\u003eO emissions in soil in two different rice cultivation modes, namely, rice planting and non-rice planting treatments. N\u003csub\u003e2\u003c/sub\u003eO emissions in the upland rice seasons were significantly higher than those in the flooded rice cultivation seasons, regardless of rice planting. Although higher rice yield was determined in UR rotation than in RR rotation, the yield-based N\u003csub\u003e2\u003c/sub\u003eO emissions were higher in the UR model than in the RR model.\u0026nbsp;These results could be attributed to the\u0026nbsp;stronger\u0026nbsp;responses of\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO emission fluxes to soil available\u0026nbsp;N in\u0026nbsp;UR rotation than in RR rotation.\u0026nbsp;The responses of\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO emission fluxes to soil ammonium NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and\u0026nbsp;DOC in\u0026nbsp;UR rotation were\u0026nbsp;stronger than those in\u0026nbsp;RR rotation. Furthermore, N\u003csub\u003e2\u003c/sub\u003eO emissions from rice-cultivated treatments were higher than those from non-rice treatments for both rice-based rotations.\u0026nbsp;The increase in\u0026nbsp;N\u003csub\u003e2\u003c/sub\u003eO emissions in rice-free treatment compared to rice-cultivated treatment could be attributed to the abundance of AOA-\u003cem\u003eamoA\u003c/em\u003e and AOB-\u003cem\u003eamoA\u003c/em\u003e genes and elevated soil mineral nitrogen content under UR rotation. While, the higher amount of N\u003csub\u003e2\u003c/sub\u003eO from rice-free treatment compared to rice-cultivated treatment under RR rotation was ascribed to the increased abundance of the \u003cem\u003enirS\u003c/em\u003e gene and the decreased abundance of the \u003cem\u003enosZ\u003c/em\u003e gene.\u0026nbsp;Our results highlight that the response of N\u003csub\u003e2\u003c/sub\u003eO emissions mainly relies on soil available N and C and critical functional genes, which should be the focus when investigating N\u003csub\u003e2\u003c/sub\u003eO mitigation measures in the future. The contribution of rice plantation to decreased field N\u003csub\u003e2\u003c/sub\u003eO emission during rice cultivation seasons should also be investigated in comparison with non-rice plantation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLastly, the writers would like to express their gratitude to Key Research and Development Project of Hubei Province for funding this study (2021BCA156).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBakken LR (1988) Dentrification under different cultivated plants: effects of soil moisture tension, nitrate concentration, and photosynthetic activity, Biol Fertil Soils 6: 271-278.\u003c/li\u003e\n\u003cli\u003eBolton H, Smith JL, Wildung RE (1990) Nitrogen mineralization potential of shrub-steppe soils with different disturbance histories. 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Sci Total Environ 811: 151402.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rice-based rotations, N2O emissions, Soil variables, Regulatory factors, Functional genes","lastPublishedDoi":"10.21203/rs.3.rs-3428312/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3428312/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims \u003c/strong\u003eAlthough the effects of upland and flooded rice cultivation on soil N\u003csub\u003e2\u003c/sub\u003eO emissions have been reported, scholars have not comparatively investigated the mechanism underlying N\u003csub\u003e2\u003c/sub\u003eO emissions during the rice cultivation seasons of rice-based rotation systems.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Herein, a two-year field experiment including two rice cultivation modes, namely, conventional upland rice–rapeseed (UR-CC) and flooded rice–rapeseed (RR-CC) rotations, was conducted to determine\u003cem\u003e \u003c/em\u003ethe effect of different rice plantation models on soil N\u003csub\u003e2\u003c/sub\u003eO emissions. Non-rice treatments (UR-NC and RR-NC) during the rice season were also implemented to confirm the effect of rice plantation or soil condition on N\u003csub\u003e2\u003c/sub\u003eO emissions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eSeasonal N\u003csub\u003e2\u003c/sub\u003eO emissions were higher in UR-CC rotation than in RR-CC rotation (1.54 ± 0.16 \u003cem\u003evs\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e0.71 ± 0.20 and 2.57 ± 0.28 \u003cem\u003evs.\u003c/em\u003e 0.76 ± 0.04 kg N ha\u003csup\u003e-1\u003c/sup\u003e for the first and following rice cultivation seasons, respectively). Also, N\u003csub\u003e2\u003c/sub\u003eO emissions were higher in UR-NC treatment than that in RR-NC treatment during both rice seasons (2.45 ± 0.07 \u003cem\u003evs\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e1.43 ± 0.35 and 3.74 ± 0.37 \u003cem\u003evs\u003c/em\u003e. 1.16 ± 0.08 kg N ha\u003csup\u003e-1\u003c/sup\u003e, respectively). The yield-based N\u003csub\u003e2\u003c/sub\u003eO emissions were higher in the UR model than in the RR model (0.21 ± 0.01 \u003cem\u003evs\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e0.10 ± 0.02 and 0.34 ± 0.03 \u003cem\u003evs.\u003c/em\u003e 0.11 ± 0.01, respectively).\u003cem\u003e \u003c/em\u003eThe responses of N\u003csub\u003e2\u003c/sub\u003eO emission fluxes to soil ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) and dissolved organic carbon (DOC) in UR rotation were stronger than those in RR rotation. Furthermore, total N\u003csub\u003e2\u003c/sub\u003eO emissions from non-rice treatments were higher than those from rice-cultivated treatments for both rice-based rotations. The increase in N\u003csub\u003e2\u003c/sub\u003eO emissions in UR-NC treatment could be attributed to the higher abundance of \u003cem\u003eamoA\u003c/em\u003e gene and elevated soil mineral nitrogen content compared to UR-CC treatment. The higher amount of N\u003csub\u003e2\u003c/sub\u003eO generated in RR-NC treatment than that in RR-CC treatment was ascribed to the increased abundance of the \u003cem\u003enirS\u003c/em\u003e gene and the decreased abundance of the \u003cem\u003enosZ\u003c/em\u003e gene. The structural equation model supported that soil moisture, temperature, available C and N, and ammonium oxidation-related functional genes explained more than 70% of the effect on soil N\u003csub\u003e2\u003c/sub\u003eO emissions in UR rotation. Meanwhile, soil moisture, temperature, available N, and denitrification-related functional genes explained 80% of the effect in RR rotation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eThese findings highlight the importance of rice plantation and their contribution to decreased field N\u003csub\u003e2\u003c/sub\u003eO emission, and suggest that soil available C, N, and critical functional genes should be considered when investigating N\u003csub\u003e2\u003c/sub\u003eO mitigation pathways during rice cultivation seasons.\u003c/p\u003e","manuscriptTitle":"Contrasting regulating effects of soil available nitrogen, carbon, and critical functional genes on soil N 2 O emissions between two rice-based rotations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-16 17:29:33","doi":"10.21203/rs.3.rs-3428312/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fdbacc3-39cb-41cb-9ffe-731db9d0f540","owner":[],"postedDate":"October 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-20T04:20:58+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-16 17:29:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3428312","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3428312","identity":"rs-3428312","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}