Elevated CO2 and Nitrogen Supply Boost N Use Efficiency and Wheat (T. aestivum cv. Yunmai) Growth and Differentiate Soil Microbial Communities Related to Ammonia Oxidization

Elevated CO2 levels (eCO2) pose challenges to wheat (Triticum aestivum L.) growth, potentially leading to a decline in quality and productivity. This study addresses the effects of two ambient CO2 concentrations (aCO2, daytime/nighttime = 410/450 ± 30 ppm and eCO2, 550/600 ± 30 ppm) and two nitrogen (N) supplements (without N supply—N0 and with 100 mg N supply as urea per kg soil—N100) on wheat (T. aestivum cv. Yunmai) growth, N accumulation, and soil microbial communities related to ammonia oxidization. The data showed that the N supply effectively mitigated the negative impacts of eCO2 on wheat growth by reducing intercellular CO2 concentrations while enhancing photosynthesis parameters. Notably, the N supply significantly increased N concentrations in wheat tissues and biomass production, thereby boosting N accumulation in seeds, shoots, and roots. eCO2 increased the agronomic efficiency of applied N (AEN) and the physiological efficiency of applied N (PEN) under N supply. Plant tissue N concentrations and accumulations are positively related to plant biomass production and soil NO3−-N. Additionally, the N supply increased the richness and evenness of the soil microbial community, particularly Nitrososphaeraceae, Nitrosospira, and Nitrosomonas, which responded differently to N availability under both aCO2 and eCO2. These results underscore the importance and complexity of optimizing N supply and eCO2 for enhancing crop tissue N accumulation and yield production as well as activating nitrification-related microbial activities for soil inorganic N availability under future global environment change scenarios.


Introduction
As CO 2 is a fundamental component of photosynthesis, its increase can initially seem beneficial, promoting faster growth and potentially higher yields in various plant species [1].This suggests a promising aspect for promoting agricultural productivity, yet the benefits are shadowed by complex challenges (IPCC, 2021) [2].Elevated CO 2 (eCO 2 ) levels (2.0 to 2.4 ppm per year since 2000) contribute to global warming, altering weather patterns and threatening food security through the potential loss of crop diversity and productivity in most regions [3].Furthermore, the nutritional quality of crop produce can be diminished under higher CO 2 conditions, impacting human health by reducing the availability of essential nutrients in staple foods [4,5].Therefore, understanding the relationship among CO 2 , plant growth, and human living necessitates a nuanced approach,

Variation in Basic Photosynthetic Characteristics
The effects of CO 2 and N supply on photosynthesis parameters during the booting stage of wheat was found to be diverse and differed between aCO 2 and eCO 2 conditions.Under aCO 2 , the application of N fertilizer significantly decreased the intercellular CO 2 concentration (Ci) (Figure 2C), which suggested that higher N availability might reduce the internal CO 2 concentration within the leaf, potentially due to increased photosynthetic activity or altered stomatal behavior.However, under eCO 2 , this effect was not observed, indicating that eCO 2 might mitigate or alter the response of Ci to N supply.There was no significant change in the net photosynthesis rate (Pn) (Figure 2A), transpiration rate (E) (Figure 2B), and stomatal conductance (Gs) (Figure 2D) under both aCO 2 and eCO 2 with N100 supply.This suggested that while N supply could influence photosynthetic parameters, its impact on these specific metrics was not significant under these tested conditions.
All the measured parameters, including Ci, Pn, E, and Gs, were significantly greater under eCO 2 than under aCO 2 for both N0 and N100 treatments (Figure 2).This indicates that eCO 2 generally enhanced wheat's photosynthetic capacity, potentially due to the direct effect of higher CO 2 levels on photosynthesis, known as the CO 2 fertilization effect.This effect can increase the net photosynthetic rate by reducing photorespiration and increasing the efficiency of the Calvin cycle.Additionally, there was no significant change in the net photosynthetic rate of N0 under eCO 2 , suggesting that even without additional N supply, eCO 2 can promote photosynthesis in wheat.Overall, these results indicated that eCO 2 enhanced various aspects of photosynthesis in wheat during the booting stage, potentially leading to increased C assimilation and growth.The differential responses to N supply under aCO 2 and eCO 2 highlighted the importance of considering both CO 2 levels and N availability when assessing plant photosynthetic performance and productivity.Lower-case letters above the bars indicate a significant difference between N supplies for the same aCO 2 treatment (a, b) or eCO 2 treatment (A,B) and between CO 2 concentrations for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil.
case letters above the bars indicate a significant difference between N supplies for the same aCO2 treatment (a, b) or eCO2 treatment (A,B) and between CO2 concentrations for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO2, atmospheric CO2; eCO2, elevated CO2; N0, no N supply; N100, 100 mg N kg −1 DW soil.A,B) and between CO 2 concentrations for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil.

Variation in Tissue N Concentrations
Under aCO 2 conditions, N100 resulted in a significant improvement in seed, shoot, and root N concentrations (Figure 3A,C,E), indicating that higher N availability enhanced N content in these tissues.However, this N supply did not significantly influence the seed, shoot, and root N accumulation (Figure 3B,D,F).These results indicate that while the concentration of N in plant tissues increased, the total amount of N that accumulated in these tissues did not change significantly.Under eCO 2 conditions, N supply significantly increased N concentrations in seeds, shoots, and roots.This suggested that eCO 2 enhanced the responsiveness of plants to N supply in terms of tissue N content.The level of N accumulation under N supply was significantly increased in shoots and roots but did not show a significant increase in seeds under eCO 2 .These results indicated that eCO 2 might alter the distribution of N within the plant, favoring accumulation in certain tissues over others.Interestingly, shoot N concentrations for the N0 treatment were significantly greater under eCO 2 than under aCO 2 , while they were similar in shoots for the N100 and also in seeds and roots for under N0 and N100, regardless of N fertilization.These findings suggested that eCO 2 might influence N uptake or internal N redistribution even in the absence of additional N supply.
Regarding N accumulation in N0, seeds exhibited significantly decreased N accumulation, and shoots exhibited increased N accumulation under eCO 2 .Roots showed no significant change.This indicated a shift in N allocation under eCO 2 , potentially favoring shoot growth at the expense of seed N accumulation when the N supply is limited.The increasing trend of N accumulation in shoots and roots and the decreasing trend in seeds under eCO 2 were further aggravated by N supply.This suggested that eCO 2 and N supply interact to influence N distribution within the plant, potentially affecting crop yield and quality.Overall, the interactive effect of N supply and eCO 2 on N concentrations was observed in both shoots and seeds (Figure 3), highlighting the complex interplay between CO 2 levels and N availability in shaping plant N dynamics and potentially impacting agricultural productivity and sustainability.

Changes in Soil pH, Organic Matter, and Concentrations of NH 4
+ and NO 3

−
Under both aCO 2 and eCO 2 , N supply led to a significant decrease in soil pH (Figure 4A).This was likely due to the acidifying effect of N fertilizers, which can release hydrogen ions into soil to decrease pH.Nitrogen supply did not result in changes in soil organic matter (Figure 4B), soil total N (Figure 4C), and soil NH 4 + -N concentrations (Figure 4E), but a significant increase in soil NO 3 − -N concentrations (Figure 4F) was noted, indicating that the N supply enhanced the availability of NO 3 − , a more mobile inorganic N form in soil.Under aCO 2 , the N supply also significantly increased the C-to-N (C/N) ratio.This suggested that while the N supply increased soil N, it might not proportionally increase soil organic matter, leading to a higher C/N ratio.Under both N0 and N100, the C/N ratio, soil NH 4 + -N, and soil NO 3 − -N were significantly lower under eCO 2 than under aCO 2 (Figure 4C-E).This indicated that eCO 2 might influence the soil N cycle, potentially reducing the availability of both ammonium and NO 3 − in soil.Contrastingly, both soil organic matter and total N exhibited significant increases under both N0 and N100, with the exception of soil organic matter under N0.This suggested that the N supply generally enhanced soil N and organic matter, but the response to eCO 2 could vary depending on the level of N supply.Overall, these results indicated that the N supply decreased soil pH, while eCO 2 decreased the C/N ratio, soil NH 4 + -N, and soil NO 3 − -N concentrations.These findings highlight the complex interactions among CO 2 levels, N supply, and soil properties, which can influence soil fertility and plant nutrition.

Nitrogen Use Efficiency
Under N100 supply, eCO 2 enhanced the agronomic efficiency of applied N (AE N ) and physiological efficiency of applied N (PE N ) compared to aCO 2 conditions, as indicated in Table 1.This suggested that eCO 2 promoted a more efficient use of applied N in terms of yield and biomass production per unit of N applied.However, eCO 2 decreased the crop recovery efficiency of applied N (RE N ) and partial factor productivity of applied N (PFP N ) compared to aCO 2 under N100 supply (Table 1).This indicated that while eCO 2 improved the utilization efficiency of applied N within the plant, it might reduce the overall recovery of applied N by the crop and the productivity per unit of N applied in terms of total biomass or yield.These findings highlight the complex interactions between CO 2 Plants 2024, 13, 2345 7 of 24 levels and N use efficiency in crops, with eCO 2 potentially altering the balance among N uptake, utilization, and productivity.B,E) and root (C,F) of 5-month-old wheat at harvest.Data are means ± SE (n = 3).Lower-case letters above the bars indicate significant differences between N supplies for the same aCO2 treatment (a, b) or eCO2 treatment (A,B) and between CO2 treatments for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO2, atmospheric CO2; eCO2, elevated CO2; N0, no N supply; N100, 100 mg N kg −1 DW soil.B,E) and root (C,F) of 5-month-old wheat at harvest.Data are means ± SE (n = 3).Lower-case letters above the bars indicate significant differences between N supplies for the same aCO 2 treatment (a, b) or eCO 2 treatment (A,B) and between CO 2 treatments for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil.A,B) and between CO2 concentrations for the same N treatment (x, y) at p < 0.05.

Nitrogen Use Efficiency
Under N100 supply, eCO2 enhanced the agronomic efficiency of applied N (AEN) and physiological efficiency of applied N (PEN) compared to aCO2 conditions, as indicated in Table 1.This suggested that eCO2 promoted a more efficient use of applied N in terms of yield and biomass production per unit of N applied.However, eCO2 decreased the crop recovery efficiency of applied N (REN) and partial factor productivity of applied N (PFPN) compared to aCO2 under N100 supply (Table 1).This indicated that while eCO2 improved the utilization efficiency of applied N within the plant, it might reduce the overall recovery of applied N by the crop and the productivity per unit of N applied in terms of total biomass or yield.These findings highlight the complex interactions between CO2 levels and N use efficiency in crops, with eCO2 potentially altering the balance among N uptake, utilization, and productivity.

Characters of Relationships between Soil Inorganic N and Plant N
Wheat total biomass production exhibited varied correlations with soil and plant tissue N concentrations under different CO 2 conditions.Under eCO 2 , wheat biomass production did not show significant correlations with soil NH 4 + -N (p = 0.20-0.24, Figure 5A), soil Plants 2024, 13, 2345 9 of 24 total N concentrations (p = 0.35-0.85, Figure 5C), or soil NO 3 − -N (p = 0.42, Figure 5B).Similarly, under aCO 2 , there was no significant correlation between wheat biomass production and shoot total N concentrations (p = 0.05, Figure 5E).However, under aCO 2 , wheat biomass production significantly positively correlated with soil NO 3 − -N (p < 0.01, Figure 5B), indicating a strong relationship between soil NO 3 − availability and biomass production.Additionally, under eCO 2 , wheat biomass production showed a significant positive correlation with shoot N concentration (p < 0.05, Figure 5E), suggesting an enhanced role of shoot N in biomass production under eCO 2 .Furthermore, wheat biomass production significantly positively correlated with seed N concentration under both aCO 2 and eCO 2 (P aCO 2 < 0.01, P eCO 2 < 0.05, Figure 5D) and root N concentration under both aCO 2 and eCO 2 (P aCO 2 < 0.01, P eCO 2 < 0.05, Figure 5F), highlighting the importance of seed and root N accumulation in biomass production across different CO 2 levels.Plant tissue total N concentrations, on the other hand, did not show significant correlations with soil total N concentrations (p = 0.10-0.75, Figure 5G-I − -N (P seed < 0.05, P root < 0.01).These suggest that while soil NO 3 − availability is important for seed and root N accumulation under aCO 2 , the relationship between plant tissue N and soil N forms is complex and varies with CO 2 levels and plant tissues.
Wheat seed, shoot, and root N accumulation exhibited positive correlations with total wheat biomass production under both aCO 2 and eCO 2 .Specifically, under aCO 2 , significant positive correlations were observed for seed (p < 0.001), shoot (p < 0.05), or root (p < 0.05) N accumulation and total biomass production (Figure 6A-C).Under eCO 2 , these correlations became even stronger, with seed (p < 0.001), shoot (p < 0.01), and root (p < 0.01) N accumulation showing highly significant positive relationships with total biomass (Figure 6A-C).The eCO 2 enhanced relationship between biomass production and shoot N accumulation suggested a more efficient utilization of N in biomass production under eCO 2 .Plant tissue total N concentrations and N accumulations were found to correlate significantly positively under both aCO 2 and eCO 2 .Under aCO 2 , significantly positive correlations were observed for seed (p < 0.01), shoot (p < 0.001), and root (p < 0.01) N concentrations with total biomass (Figure 6D-F).Under eCO 2 , these correlations persisted, with seed (p < 0.05), shoot (p < 0.001), and root (p < 0.001) N concentrations showing significantly positive relationships with total biomass (Figure 6D-F).However, wheat tissue N accumulation did not show significant correlations with soil NH 4 + -N (p = 0.10-0.51, Figure 6G-I), soil total N concentrations (p = 0.19-0.74, Figure 6M-O), or soil NO 3 − -N (p = 0.08-0.64, Figure 6J-L) under both aCO 2 and eCO 2 .Soil NO 3 − -N was found to correlate significantly and positively with seed and root N accumulation (P seed < 0.01, P root < 0.01, Figure 6J,L), indicating a specific relationship between soil NO 3 − -N availability and N accumulation in these plant tissues.

Soil Microbial Community Composition and Species Abundance
The differences in the soil microbial community between different treatments were assessed using Illumina MiSeq sequencing of the V3-V4 region of bacterial 16S rRNA gene.These Illumina MiSeq sequencing assays were performed by followed by the instructions from the facility of an Il-559 lumina, San Diego, CA, USA.A total of 516,804 valid sequences were obtained from the soil sample, with an average of 43,067 individual sample sequence counts per sample.

Soil Microbial Community Composition and Species Abundance
The differences in the soil microbial community between different treatments were assessed using Illumina MiSeq sequencing of the V3-V4 region of bacterial 16S rRNA Alpha diversity is an ecological metric that assesses the variety and even distribution of taxonomic groups within a single sample [32].Elevated CO 2 did not significantly affect the Sobs index, Shannon index, or Simpson index under both N0 and N100, while the Ace index had a decreasing trend.Under both aCO 2 and eCO 2 , N100 significantly increased the Shannon index but decreased the Simpson index (Figure 7C).N100 significantly influenced N cycling microorganisms (p = 0.002), while the influence of eCO 2 on communities was higher under N100 than under N0 (Figure 7B).We performed redundancy analysis (RDA) to show the correlation between samples under different treatments, environmental factors, and soil N cycling microorganisms.The RDA results (RDA1 = 81.92%,RDA2 = 4.65%) showed that soil pH was the most influential factor on soil N cycling microorganisms followed by NH 4 + -N and NO 3 − -N (Figure 7A).Under both aCO 2 and eCO 2 , the N supply notably increased the relative abundance of unclassified_g_Nitrosomonas and uncultured_bacterium_g_Nitrosospira at the species level.eCO 2 treatment notably decreased metagenome_g_Nitrosomonas and increased Frankia_sp._g_Frankia under N0 (Figure 7D).
sample sequence counts per sample.
Alpha diversity is an ecological metric that assesses the variety and even distribution of taxonomic groups within a single sample [32].Elevated CO2 did not significantly affect the Sobs index, Shannon index, or Simpson index under both N0 and N100, while the Ace index had a decreasing trend.Under both aCO2 and eCO2, N100 significantly increased the Shannon index but decreased the Simpson index (Figure 7C).N100 significantly influenced N cycling microorganisms (p = 0.002), while the influence of eCO2 on communities was higher under N100 than under N0 (Figure 7B).We performed redundancy analysis (RDA) to show the correlation between samples under different treatments, environmental factors, and soil N cycling microorganisms.The RDA results (RDA1 = 81.92%,RDA2 = 4.65%) showed that soil pH was the most influential factor on soil N cycling microorganisms followed by NH4 + -N and NO3 − -N (Figure 7A).Under both aCO2 and eCO2, the N supply notably increased the relative abundance of unclassified_g_Nitrosomonas and un-cultured_bacterium_g_Nitrosospira at the species level.eCO2 treatment notably decreased metagenome_g_Nitrosomonas and increased Frankia_sp._g_Frankia under N0 (Figure 7D).
Significant differences among four treatments were observed in the relative abundance of soil N cycling microorganisms at the genus level of the 3 most abundant genera (Figure 8A).These 3 genera were dominating the communities for N cycling in soil from all treatments.Nitrososphaeraceae was the main microbial genus present at N0 under both aCO2 and eCO2 (p < 0.001-0.01).Nitrosospira and Nitrosomonas in the soil under N100 treatment were more abundant compared to N0 under both aCO2 and eCO2 (p < 0.001-0.05)(Figure 8B).These results meant that the N supply was the main factor influencing soil microbial communities.Significant differences among four treatments were observed in the relative abundance of soil N cycling microorganisms at the genus level of the 3 most abundant genera (Figure 8A).These 3 genera were dominating the communities for N cycling in soil from all treatments.Nitrososphaeraceae was the main microbial genus present at N0 under both aCO 2 and eCO 2 (p < 0.001-0.01).Nitrosospira and Nitrosomonas in the soil under N100 treatment were more abundant compared to N0 under both aCO 2 and eCO 2 (p < 0.001-0.05)(Figure 8B).These results meant that the N supply was the main factor influencing soil microbial communities.

Nitrogen Supply Generally Rebalanced the Effects of eCO 2 on Wheat Growth
Elevated CO 2 (eCO 2 ) did not significantly increase wheat biomass production; in fact, it somewhat reduced the growth biomass under N0 (Figure 1B-E).This result was Plants 2024, 13, 2345 14 of 24 in a disagreement with results from other studies [5,9,33] because a good normal biomass production needs an element match-up of C and N stoichiometry [34] and also highly depends on cultivars as two out five wheat cultivars had a lower shoot dry weight production under 800 ppm CO 2 than under 400 ppm CO 2 [9].Compared to aCO 2 (400 ppm), an NH 4 + -tolerant pea (Pisum sativum L. cv.snap pea) plant under eCO 2 (800 ppm) had to cope with an unbalanced C-to-N ratio due to a limited C sink strength and/or constrains in leaf N content in order to promote photosynthetic efficiency and C allocation into sugars (glucose and sucrose) under moderate (2.5 mM) or high (10 mM) NH 4 + supplement [35].Moreover, a meta-analysis of 22 studies showed that an enhanced aboveground biomass production and grain yield was only observed when 18 barley genotypes were cultivated under a combination of eCO 2 (651-720 ppm) and a higher N rate (151-200 kg ha −1 ) [36].In addition, a review on studies between 1986 and 2022 (n = 20) showed that strawberry yield was also decreased under 1588 ± 582 CO 2 ppm than under aCO 2 [37].Nevertheless, the N supply and CO 2 treatment did not significantly influence the harvest index (Figure 1F).In general, the effect of CO 2 fertilization on photosynthesis had been significantly reduced globally during the last four decades (1982 to 2015) [38], although the study's key conclusion was not robust mainly due to negligence regarding the role of the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase [39].
When the environmental CO 2 concentration increased from 372 ppm to 605 ppm, the response for wheat yield indicated that at about 600 ppm CO 2 , the stimulation of yield tended to stabilize, suggesting the existence of a plateau phase, beyond which the yield might not be significantly increased [40].Furthermore, the response of wheat to the increase in CO 2 concentration is significantly related to the productivity of the growth site [41].In systems with a lower productivity, the relative stimulation effect of CO 2 on yield was stronger [42].Wheat biomass production was significantly increased by N100 supply under both atmospheric CO 2 (aCO 2 ) and eCO 2 (Figure 1B-E), which was consistent with results from other studies [43,44].This suggests that N generally boosts wheat biomass by supporting essential physiological processes, whereas eCO 2 decreases biomass by disrupting nutrient balance and water relations or limitations.In the latter case, our results indeed showed that eCO 2 significantly decreased the water use efficiency at the leaf scale (WUEi) under N0 (Figure S2).As a result, the fertilization effect of eCO 2 on wheat biomass production can only be achieved when N and/or water supplementation are adequate.This illustrates the complex interplay between these factors in plant growth.

Elevated CO 2 Changed the Decreasing Trend of N Supply on Photosynthesis Parameters
The effects of N fertilizer and CO 2 concentration on the photosynthetic parameters of wheat were different.The stomatal conductance and net photosynthetic rate were lower at N100 than at N0 under aCO 2 .In addition, the intercellular CO 2 concentration was significantly decreased at N100 under aCO 2 , but the photosynthesis parameters under eCO 2 were not significantly affected by N supply during the booting stage (Figure 2).Nitrogen is essential for plant photosynthesis because its availability significantly influences the efficiency of the photosynthetic process and, by extension, the overall growth and performance of plants [45].A study performed by Wu et al. also showed that N application had significant decrease on the intercellular CO 2 concentration of wheat at the booting stage [46].Applying excessive N fertilizer may lead to an over-absorption of N by plants, which can increase the chlorophyll content in wheat, enhance the rate of photosynthesis, and thus increase the rate of CO 2 absorption, resulting in a reduction of leaf intercellular CO 2 concentrations [46][47][48].Elevated CO 2 significantly increased the photosynthetic parameters under both N0 and N100 (Figure 2).A study measured photosynthesis in winter wheat and rice under different CO 2 levels and also found that eCO 2 generally increased net photosynthesis by 10-40% at different development stages [49,50].The increase in CO 2 concentration to some extent compensates for the restrictive effect of N fertilizer application on the formation during the booting stage.

Nitrogen Supply Mitigated the Reduction of Seed N Accumulation with eCO 2 Treatment
Elevated CO 2 increased N concentrations in both shoots and roots of wheat but decreased the seed N concentration under N0 (Figures 3A,C,E and 5J,M).N100 enhanced such increasing trends in seed and root N concentrations, while shoot N concentrations were decreased under eCO 2 (Figure 3A,C,E).These results were similar to Andrea et al.'s study demonstrating that eCO 2 reduced the N content and nutritional quality of wheat and other crops [51,52].Elevated CO 2 significantly decreased seed N accumulation under N0, while this trend was stopped under N100 (Figure 3B).These results might explain why eCO 2 and increased N fertilizer significantly increased the number of tillers (Figure S1) and aboveground dry biomass of wheat (Figure 1) but reduced the leaf chlorophyll content (as a proxy for plant N content) [53].However, of note, N accumulation in shoots and roots was much higher at eCO 2 than at aCO 2 under N100 (Figure 3D,F).Studies have focused on the regulation of N allocation in plants by N application rates.During the period from flowering to maturity, the N translocation in individual plant aboveground organs decreased with the downward spatial position [54].Increased N application could effectively enhance the absorption of N, delaying leaf senescence and death (Figures 5O and 6L).It is hence important to fertilize at the proper stage of wheat.

Elevated CO 2 Decreased Active Nitrogen under Both N0 and N100
The pH decreased because of nitrification ( 2NH ) under both aCO 2 and eCO 2 , which is important for plant growth [55] (Figure 4A).The concentrations of NH 4 + and NO 3 − under eCO 2 were significantly decreased under both N0 and N100, while soil total N and C/N under aCO 2 and eCO 2 did not significantly change under both N0 and N100 (Figure 4C,E,F).The increase in CO 2 concentration could increase the biomass in the upper part of the crop, thereby increasing the crop's demand for N [56].The average soil total N value is 0.9 g kg −1 [57], and it was barren compared to the soil used in this study (soil total N = 0.53 g kg −1 ).Studies have shown that an increased CO 2 concentration can promote the growth and development of crop root systems, expand the distribution of root systems in soil, and increase the absorption of N [58].Because the concentration of CO 2 is much higher (about 10-50 times) in soil than in the atmosphere, the increase in the CO 2 concentration in the atmosphere has almost no direct impact on soil microorganisms [59].Instead, it has an indirect effect on soil microorganisms by affecting the root secretions and falling objects of the crop, further affecting soil N conversion [60].

Elevated CO 2 Increased N Use Efficiency for Plant Growth but Not for Yield Production under N100
Research has shown that N use efficiency is a critical factor in agricultural production and environmental sustainability [61].The concept of N use efficiency is defined as the flux ratio between plant dry matter production and N uptake, which is essential for understanding plant growth strategies and ecosystem productivity [62].The physiological efficiency of applied N (PE N ) indicates the amount of biomass produced per unit of N absorbed by the plant [63], while the partial factor productivity of applied N (PFP N ) reflects the yield increase per unit of N input [64], showing the marginal return on N use [65].The crop recovery efficiency of applied N (RE N , [66]) is an indicator of how much of the N input is effectively utilized by the crop, while AE N (agronomic efficiency of applied N) is often used to evaluate the effectiveness of N fertilizers [67].Elevated CO 2 decreased RE N and PFP N and increased AE N and PE N under N100 in this study (Table 1).These results were supported by the N concentrations and accumulations (Figures 1, 3, 5 and 6).This could mean an increase in the growth rate and overall biomass of plants in eCO 2 environments, even if the plants are not grown to harvest grains or produce high yields.Elevated CO 2 generally results in increased grain production but lower grain quality, particularly with regard to N concentration and protein content, which can lead to increased "hidden hunger" problems [68].The free amino acid and protein contents of cereals grown under eCO 2 conditions are expected to decrease significantly [42].The availability of N limited the effect of CO 2 fertilization; that is, eCO 2 increased plant biomass more significantly when the supply of N was sufficient [52].Total biomass is related positively to seed, shoot, and root N concentration and accumulation under both aCO 2 and eCO 2 (only eCO 2 for shoot N concentration) (Figures 5D-F and 6A-C).Soil NO 3 − -N is related positively to seed and root N concentration and accumulation under aCO 2 (Figure 5M,O).These results were similar to common eCO 2 results indicating an increase of grain yield but a decrease of grain quality, particularly in N concentration and protein content [42,68].
3.6.Influence of eCO 2 and N100 on the Relative Abundance and Structure of N Cycling Microorganisms Nitrogen supply led to the decreasing pH, which is the most important factor for microbial community composition in this study (Figures 4A and 7A).Soil acidification is a critically topic soil issue, and it has also been demonstrated that soil acidification significantly influenced plant diversity, species richness, and the occurrence of species [69].Previous research indicates that soil acidification can result from N deposition, acid rain, and continuous cropping practice [70][71][72][73].Nutrient-induced changes in soil pH are a primary driver controlling diversity-function relationships [74].Although eCO 2 significantly influenced plant production, it did not significantly affect soil N cycling microorganisms (Figures 1 and 7B,C).A study has shown that eCO 2 had no significant effect on nitrification, while AOA communities are more responsive to elevated temperature than AOB communities [75].N100 significantly increased the richness and evenness of the community and the relative abundance of Nitrosomonas and Nitrosospira (Figure 7B-D).Nitrososphaeraceae was significantly higher at N0 than at N100 under both aCO 2 and eCO 2 , while contrasting results were obtained for Nitrosomonas and Nitrosospira (Figure 8B).Different N requirements are noted between archaea and bacteria involved in N oxidation; for example, the requirements for Nitrososphaeraceae (archaea) versus Nitrosomonas and Nitrosospira (bacteria) are influenced by their distinct ecological roles, metabolic capacities, and evolutionary adaptations [76].AOA communities often thrive in environments with low ammonia concentrations and can tolerate a wider range of temperatures and pH levels, while AOB communities typically prefer higher ammonia concentrations and may be more sensitive to extreme environmental conditions [77].These may be the reason why AOA and AOB communities differentiate beneath soil niches under different N supplies and eCO 2 levels.

Description of the Experiment Site
The experiment site is located in the National Monitoring Base of Purple Soil Fertility and Fertilizer Effect (29 • 48 ′ N, 106 • 24 ′ E, 266.3 m above sea level) on the Beibei campus of Southwest University, Chongqing, China, which is located within the purple hilly region with a subtropical monsoon climate.Over the past three decades, the mean annual sunshine was 1276.7 h, the mean annual precipitation was 1145.5 mm, and the mean annual temperature was 18.4 • C.During the experiment period, the atmospheric CO 2 (aCO 2 ) concentration was ~415 ppm at this experiment site.The soil is classified as a purple soil (Eutric Regosol, FAO Soil Classification System), which has evolved from purple mud and shale of the Jurassic Shaximiao Formation [78] and exhibits the following basic chemical properties: pH 7.4 (1:2.5 W/V, soil/water), 9.00 g kg −1 organic matter, 0.53 g kg −1 total N, 7.81 mg kg −1 NH 4 + , and 16.47 mg kg −1 NO 3 − .

Design and Description of Custom-Built Chambers
The experiment was carried out in 6 identical enclosed gas chambers (length × width × height = 1.5 m × 1.0 m × 2.5 m), which were made of a 10 mm thickness steel frame covered with transparent glass (90% light transmission rate) (Yutao Glass Company, Chongqing, China).The gas flow solenoid valve (AirTAC (China)Co., Ltd., Ningbo, China) was connected to the metal cylinder containing pure CO 2 as the gas source.Each chamber had two air pumps (suction and intake), and the excess CO 2 and water vapor were balanced with 1 M NaOH solution and anhydrous CaCl 2 in the chamber, respectively.A hanging air conditioner (Gree, Zhuhai Gree company, Zhuhai, China) was installed on the top of the chamber to regulate the air temperature.An atmospheric light, temperature, and humidity sensor (Jingxun Electronic Technology, Weihai, China) and CO 2 concentration detector (infrared CO 2 sensor module B-530, ELT SENSOR Corp, Bucheon-si, Republic of Korea) were installed at the middle of the chamber.All these devices were deployed using fully automatic control device (DSS-QZD, Qingdao Shengsen Numerical Control Technology Institute, Qingdao, China).The whole system can automatically control the temperature, humidity, and CO 2 concentration inside and outside the glass chamber and ensure that the CO 2 concentration was maintained as required by the experiment in the chamber [30].

Designs of Experiment and Preparation of Materials
Based on the field-detected CO 2 concentration, we set up two CO 2 concentration treatments: (1) atmospheric CO 2 (aCO 2 , 410 ppm during daytime/460 ppm at night) and (2) eCO 2 (eCO 2 , 550 ppm during daytime/610 ppm at night).The time of day and night for CO 2 treatment varied with the local sunrise and sunset and also with seasons.
Wheat seeds (T.aestivum cv.Yunmai) were sterilized with a 6% (v/v) hydrogen peroxide and germinated on sterile filter paper [79].Germinated wheat seeds were sown in plastic pots (diameter = 22 cm, height = 20 cm, each containing 5 kg soil), and 4 uniform seedlings were grown inside the growth chambers until they reached the harvest stage (5 months old).
Along with the addition of P (100 mg P kg −1 DW soil, Ca(H 2 PO 4 ) 2 ) and K (126 mg K kg −1 DW soil, K 2 SO 4 ), two N fertilization treatments were also applied as follows: (1) no N supply (N0) and ( 2) 100 mg N kg −1 DW soil (N100).As a result, the experiment had four treatments (two CO 2 levels and two N fertilization rates) for a total of 12 pots (each treatment had three pots or replicates).All fertilizers were thoroughly mixed with the soil before wheat planting.The potting preparation, row spacing, N supply, and irrigation followed common cultivation practices in the local area, and no pesticides or fungicides were used.To ensure a similar environment for all plants, the potted plants in the growth chamber were rotated once per week.Adequate irrigation was provided to maintain soil moisture at ~70 ± 5% of field capacity.

Measurement of Photosynthetic Parameters
During the grain booting stage of wheat, photosynthetic parameters were measured.Wheat plants with similar growth were selected, and the flag leaves of their main stem were used for measuring photosynthetic parameters.These measurements were conducted between 8:30 to 11:30 a.m. in sunny morning of 9 April 2019 using a Li-6400XT portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA) equipped with an internal red-blue light source.The light intensity was set to 1000 µmol m −2 s −1 .The CO 2 concentration in the reference chamber was set to 410 µmol mol −1 for the N0 and N100 treatments under aCO 2 and 550 µmol mol −1 for the N0 and N100 treatments under eCO 2 .The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO 2 concentration (Ci), and transpiration rate (E) were recorded, respectively.
The formula for calculating instantaneous water use efficiency at the leaf scale (WUEi) is given by [80,81]:

Preparation of Plant and Soil Samples
Plant and soil samples were collected at wheat harvest (5 months old) as depicted in Figure S3 by individuals wearing disposable gloves to avoid contamination.Plant height measurements were conducted using a tape measure from the ground to the top of the spike (excluding awns) and recorded in centimeters immediately prior to destructive sampling.Plants were further divided into roots, stems, leaves, and ears.Plant samples were dried in an oven at 70 • C for 72 h.The ears were threshed, and the number of grains per plant was also documented.The dry weight of seed, shoot, and root biomass production was recorded.Harvest index was determined using the established formula as previously described in the literature [82,83]: Seed production Shoot biomass production A total of 10 soil cores from different locations within the same pot were collected to create a composite sample for minimizing spatial variability.Any plant material or debris was removed from soil samples.The collected soil samples were packed in sterile Ziplock bags, transported to the laboratory in a portable refrigerator (−18 • C), and stored at −80 • C for soil DNA extraction.A portion of soil samples were ground through 2 mm and 0.25 mm sieves and air-dried for soil physical and chemical properties analysis.

Determination of Plant and Soil Chemical Characters
Using a LE438 composite electrode meter (Mettler Toledo Instrument Co., Ltd., Shanghai, China), soil pH was determined with a soil to water ratio of 1:2.5 (W/V).Determination of soil organic matter was performed using the K 2 Cr 2 O 7 external heating method, and soil total N was assessed using the Kjeldahl method.Soil soluble inorganic N (NH 4 + -N and NO 3 − -N) was measured using colorimetric methods with a spectrometer (UV-1800, AOE Instruments, Shanghai, China) at 625 nm (only the same amount of reagent but no soil leaching solution at 625 nm as a blank control) [84] and 275 nm (deionized water at 220 nm as blank control) [85], respectively.Plant total N was determined using the H 2 SO 4 -H 2 O 2 digestion and distillation method, which involved boiling the test solution with a mixture of sulfuric acid and hydrogen peroxide.All the parameters were determined according to relevant methodologies [86].

Calculations of Nitrogen Use Efficiency
RE N -Crop recovery efficiency of applied N (g increase in N uptake per g N applied): RE N (%) = (U N − U 0 )/F N PE N -Physiological efficiency of applied N (g yield increase per g increase in N uptake from fertilizer): PE N (g g −1 ) = (Y N − Y 0 )/(U N − U 0 ) PFP N -Partial factor productivity of applied N (often simply called N use efficiency or NUE) (g harvest product per g N applied): PFP N (g g −1 ) = Y N /F N AE N -Agronomic efficiency of applied N (g yield increase per g N applied): AE N (g g −1 ) = (Y N − Y 0 )/F N The meanings of these short terms were as follows: F N -amount of (fertilizer) N applied (g kg −1 DW); Y N -crop yield with applied N (g kg −1 DW); Y 0 -crop yield (g kg −1 DW) in a control treatment with no N; U N -total plant N uptake in aboveground biomass at maturity or harvest (g kg −1 DW) in a plot that received N; U 0 -the total N uptake in aboveground biomass at maturity or harvest (g kg −1 DW) in a place that received no N.

Analysis of Soil Bacterial and Archaeal Community Based on Illumina Sequencing
Total DNA of soil microorganisms was extracted from 2 mL sludge using a FastDNA ® SPIN Kit for Soil (MP Biomedicals, LLC, Irvine, CA, USA).The specific operation was strictly performed in accordance with the kit's instructions.The extracted total microbial DNA was stored in a refrigerator at −20 • C for future assays.The extracted genomic DNA was assessed using 1% agarose gel electrophoresis (Bio-Rad Inc., Hercules, CA, USA) and a NanoDrop-2000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA).Three replicates were extracted from each composite soil sample, and the extracted DNA was pooled together.Each treatment had three composite DNA samples.The bacterial community composition of rhizosphere soil was analyzed using high-throughput amplification sequencing.A forward primer 515FmodF (GTGYCAGCMGCCGCGGTAA) and a reverse primer 806RmodR (GGACTACNVGGGTWTCTAAT) were used for bacterial and archaeal 16S rRNA gene PCR amplification of the V4 region and then sequenced by following the assay instructions of Illumina MiSeq sequencing technology (Il-559 Illumina, San Diego, CA, USA).The operational taxonomic units (OTUs) were classified by Usearch (v7.1) with a 97% sequence similarity threshold.The microbial community structure and relative abundance were obtained by OTUs with an online platform, namely, the Majorbio Cloud (https://cloud.majorbio.com/(accessed on 20 June 2024)).

Statistical Analysis
SPSS 19.0 (SPSS Inc., Chicago, IL, USA) was used to analyze differences between different treatments using one-way ANOVA.Data (means ± SE, n = 3) were compared using the Duncan's multiple range test at p < 0.05 level.GraphPad Prism (GraphPad Software 8.0.2) was used to analyze characters of relationships.Alpha diversity analyses were completed utilizing the QIIME2 platform, followed by a visualization of all the results with graphical representations generated using mothur (version v.1.30.2 https: //mothur.org/wiki/calculators/(accessed on 21 June 2024) [87].Variations in the relative abundances of bacterial species were described in a heat map that was generated using the vegan package in the 3.3.1 R version.Principal coordinate analysis (PCoA) was achieved based on the Bray-Curtis distance matrix derived from the OTU information of each sample using R version 3.3.1 [88].Redundancy analysis (RDA) was performed to clarify the relationships between sample distributions and environmental factors [89].First, detrended correspondence analysis (DCA) was applied to the species-sample data matrix, derived from 97% OTU similarity, to determine the gradient length.Based on the DCA results, the bioenv function was then used to assess the maximum Pearson correlation coefficient between environmental factors and community distributions for identifying a subset of significantly environmental factors.The species distribution table and the complete set of environmental factors or the identified subset were then subjected to RDA.The significance of the RDA results was evaluated using a permutation test analogous to ANOVA, implemented using the vegan package of R version 3.3.1.This package also facilitated the RDA analysis and graphical representation.The Kruskal-Wallis H test and the Student's t test were performed using the stats package in R version 3.3.1.Briefly, the Kruskal-Wallis H test, a non-parametric method, was employed to assess differences among multiple groups of samples, while the Student's t test for equal variances was utilized to evaluate whether the means of two sample groups with homoscedasticity (equal variances) were identical.These analyses were performed to determine significant differences in species distribution between the groups and to adjust the p values using appropriate multiple correction methods.

FFigure 1 .Figure 1 .
Figure 1.Effects of CO2 and N supply on 5-month-old wheat at harvest.(A) Plant height; (B) Seed number; (C) Seed production; (D) Shoot (stem and leaf) biomass production; (E) Root biomass production; (F) Harvest index = seed production/shoot biomass.Data are means ± SE (n = 3).Lower-Figure 1. Effects of CO 2 and N supply on 5-month-old wheat at harvest.(A) Plant height; (B) Seed number; (C) Seed production; (D) Shoot (stem and leaf) biomass production; (E) Root biomass production; (F) Harvest index = seed production/shoot biomass.Data are means ± SE (n = 3).Lower-case letters above the bars indicate a significant difference between N supplies for the same aCO 2 treatment (a, b) or eCO 2 treatment (A,B) and between CO 2 concentrations for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil.

Figure 2 .
Figure 2. Effects of CO2 and N supply on photosynthesis parameters during the booting stage of 5month-old wheat.(A) Net photosynthetic rate; (B) Transpiration rate; (C) Intercellular CO2 concentration; (D) Stomatal conductance.Data are means ± SE (n = 3).Lower-case letters above the bars indicate a significant difference between N supplies for the same aCO2 treatment (a, b) or eCO2 treatment (A,B) and between CO2 concentrations for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO2, atmospheric CO2; eCO2, elevated CO2; N0, no N supply; N100, 100 mg N kg −1 DW soil.

Figure 2 .
Figure 2. Effects of CO 2 and N supply on photosynthesis parameters during the booting stage of 5-month-old wheat.(A) Net photosynthetic rate; (B) Transpiration rate; (C) Intercellular CO 2 concentration; (D) Stomatal conductance.Data are means ± SE (n = 3).Lower-case letters above the bars indicate a significant difference between N supplies for the same aCO 2 treatment (a, b) or eCO 2 treatment (A,B) and between CO 2 concentrations for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil.

Figure 3 .
Figure 3. Effects of CO2 and N supply on tissue N concentrations and accumulations in seed (A,D), shoot (stem and leaf) (B,E) and root (C,F) of 5-month-old wheat at harvest.Data are means ± SE (n = 3).Lower-case letters above the bars indicate significant differences between N supplies for the same aCO2 treatment (a, b) or eCO2 treatment (A,B) and between CO2 treatments for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO2, atmospheric CO2; eCO2, elevated CO2; N0, no N supply; N100, 100 mg N kg −1 DW soil.

Figure 3 .
Figure 3. Effects of CO 2 and N supply on tissue N concentrations and accumulations in seed (A,D), shoot (stem and leaf) (B,E) and root (C,F) of 5-month-old wheat at harvest.Data are means ± SE (n = 3).Lower-case letters above the bars indicate significant differences between N supplies for the same aCO 2 treatment (a, b) or eCO 2 treatment (A,B) and between CO 2 treatments for the same N treatment (x, y) at p < 0.05.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil.

Figure 4 .
Figure 4. Effects of CO 2 and N supply on (A) Soil pH; (B) Soil organic matter; (C) Soil total nitrogen; (D) C/N; (E) NH 4 + -N; (F) NO 3 − -N at harvest of 5-month-old wheat.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil.Data are means ± SE (n = 3).Lower-case letters above the bars indicate significant differences between N supply for the same aCO 2 treatment (a, b) or eCO 2 treatment (A,B) and between CO 2 concentrations for the same N treatment (x, y) at p < 0.05.

Figure 5 .
Figure 5. Relationships between soil NH4 + -N (A), soil NO3 − -N (B) or soil total nitrogen (C) and plant total biomass production (A-C); between tissue N concentrations in seed (D), shoot (E) or root (F) and plant total biomass production (D-F); between tissue N concentrations in seed (G), shoot (H) or root (I) and soil total nitrogen (G-I); between tissue N concentrations in seed (J), shoot (K) or root (L) and soil NH4 + -N (J-L); between tissue N concentrations in seed (M), shoot (N) or root (O) and soil NO3 − -N (M-O) in 5-month-old wheat grown under atmospheric CO2 (aCO2) and elevated CO2 (eCO2).Open and closed circles represent data under aCO2 and eCO2, respectively.Regressions are shown for aCO2 (dotted lines) and for eCO2 (solid lines) treatments, n = 6.

Figure 5 .
Figure 5. Relationships between soil NH 4 + -N (A), soil NO 3 − -N (B) or soil total nitrogen (C) and plant total biomass production (A-C); between tissue N concentrations in seed (D), shoot (E) or root (F) and plant total biomass production (D-F); between tissue N concentrations in seed (G), shoot (H) or root (I) and soil total nitrogen (G-I); between tissue N concentrations in seed (J), shoot (K) or root (L) and soil NH 4 + -N (J-L); between tissue N concentrations in seed (M), shoot (N) or root (O) and soil NO 3 − -N (M-O) in 5-month-old wheat grown under atmospheric CO 2 (aCO 2 ) and elevated CO 2 (eCO 2 ).Open and closed circles represent data under aCO 2 and eCO 2 , respectively.Regressions are shown for aCO 2 (dotted lines) and for eCO 2 (solid lines) treatments, n = 6.

Figure 6 .
Figure 6.Relationships between tissue N accumulations in seed (A), shoot (B) or root (C) and plant total biomass production (A-C); between tissue N concentrations in seed (D), shoot (E) or root (F) and tissue N accumulations in seed (D), shoot (E) or root (F); between tissue N accumulations in seed (G), shoot (H) or root (I) and soil NH4 + -N (G-I); between tissue N accumulations in seed (J), shoot (K) or root (L) and soil NO3 − -N (J-L); between tissue N accumulations in seed (M), shoot (N) or root (O) and soil total nitrogen (M-O) in 5-month-old wheat grown under atmospheric CO2 (aCO2) and elevated CO2 (eCO2).Open and closed circles represent data under aCO2 and eCO2, respectively.Regressions are shown for aCO2 (dotted lines) and for eCO2 (solid lines) treatments, n = 6.

Figure 6 .
Figure 6.Relationships between tissue N accumulations in seed (A), shoot (B) or root (C) and plant total biomass production (A-C); between tissue N concentrations in seed (D), shoot (E) or root (F) and tissue N accumulations in seed (D), shoot (E) or root (F); between tissue N accumulations in seed (G), shoot (H) or root (I) and soil NH 4 + -N (G-I); between tissue N accumulations in seed (J), shoot (K) or root (L) and soil NO 3 − -N (J-L); between tissue N accumulations in seed (M), shoot (N) or root (O) and soil total nitrogen (M-O) in 5-month-old wheat grown under atmospheric CO 2 (aCO 2 ) and elevated CO 2 (eCO 2 ).Open and closed circles represent data under aCO 2 and eCO 2 , respectively.Regressions are shown for aCO 2 (dotted lines) and for eCO 2 (solid lines) treatments, n = 6.

Figure 7 .Figure 7 .
Figure 7. Transition of soil microbes under different CO2 levels and N supply.(A) Redundancy analysis (RDA) plot showing the relationship between environmental factors and microbial community structure at the OTU level; (B) Principal coordinate analysis (PCoA) of microbial community composition at the OTU level; (C) Sobs, Shannon, Simpson, and Ace microbial diversity indexes at Figure 7. Transition of soil microbes under different CO 2 levels and N supply.(A) Redundancy analysis (RDA) plot showing the relationship between environmental factors and microbial community structure at the OTU level; (B) Principal coordinate analysis (PCoA) of microbial community composition at the OTU level; (C) Sobs, Shannon, Simpson, and Ace microbial diversity indexes at the OTU level.Data are means ± SE (n = 3).Lower-case letters above the bars indicate significant differences between different N supply treatments for the same CO 2 treatment (a, b) and between different CO 2 concentrations for the same N treatment (x, y) at p < 0.05; (D) Heat map shows the relative abundance of the microbial community at the species level.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil.

Plants 2024 ,
13,  x FOR PEER REVIEW 13 of 25 the OTU level.Data are means ± SE (n = 3).Lower-case letters above the bars indicate significant differences between different N supply treatments for the same CO2 treatment (a, b) and between different CO2 concentrations for the same N treatment (x, y) at p < 0.05; (D) Heat map shows the relative abundance of the microbial community at the species level.Abbreviations: aCO2, atmospheric CO2; eCO2, elevated CO2; N0, no N supply; N100, 100 mg N kg −1 DW soil.

Figure 8 .
Figure 8. Kruskal-Wallis H test (A) and Student's t-test (B) of the relative abundance of the three most abundant genera.Extended error bar plot showing the most abundant genera that had significant differences between the treatments.Positive differences in mean relative abundance indicate a genus overrepresented in the soil of the treatment, while negative differences indicate greater abundance in the soil of the treatment.Abbreviations: aCO2, atmospheric CO2; eCO2, elevated CO2; N0, no N supply; N100, 100 mg N kg −1 DW soil; *, **, *** were considered statistically significant for p < 0.05, p < 0.01, p < 0.001, respectively.

Figure 8 .
Figure 8. Kruskal-Wallis H test (A) and Student's t-test (B) of the relative abundance of the three most abundant genera.Extended error bar plot showing the most abundant genera that had significant differences between the treatments.Positive differences in mean relative abundance indicate a genus overrepresented in the soil of the treatment, while negative differences indicate greater abundance in the soil of the treatment.Abbreviations: aCO 2 , atmospheric CO 2 ; eCO 2 , elevated CO 2 ; N0, no N supply; N100, 100 mg N kg −1 DW soil; *, **, *** were considered statistically significant for p < 0.05, p < 0.01, p < 0.001, respectively.

Table 1 .
Agronomic indices of N use efficiency.Abbreviations: aCO 2 , ambient CO 2 ; eCO 2 , elevated CO 2 .Data are means ± SE (n = 3).Lower-case letters above the bars indicate a significant difference between CO 2 concentrations in the same N treatment (x, y) at p < 0.05.
• E is the transpiration rate (µmol H 2 O m −2 s −1 ); and • WUE i is the instantaneous water use efficiency at the leaf scale (µmol CO 2 µmol −1 H 2 O).