Growth Responses of Agropyron Mongolicum Keng to Nitrogen Addition Is Linked to Root Morphological Traits and Nitrogen-Use Efficiency.


 Background and aimsNitrogen (N) is the primary limiting factors for plant growth and development, and increasingly N deposition alters plant composition, consequently affecting ecosystem function have been widely acknowledged. However, the effects of N fertilization on native species in desert grassland ecosystem and underlying mechanisms of these effects are still poorly understood. This study was conducted to examine the growth response of Agropyron mongolicum keng to N addition and potential mechanisms underlying this effect.MethodsA.mongolicum Keng was subjected to five N addition levels (0, 0.8,1.6, 2.4, and 4.0g N m−2 yr−1) for six months under greenhouse conditions. A combination of linear and structural equation modelling was used to examine growth response of A.mongolicum Keng to N addition and test whether its response related to root morphological traits and N-use efficiency.ResultsGrowth responses of A. mongolicum Keng to increasing N addition appeared a unimodal-shaped with a N saturation threshold at 3.2g N m-1 yr-1. Its response closely related to the root surface area, volume, length, and forks number, N uptake and utilization efficiency. Besides, N-induced changes in soil available nutrient have an indirect impaction biomass of A. mongolicum Keng via regulation of root morphological traits and N-use efficiency.ConclusionsThese findings highlight the sensibility of A. mongolicum Keng to N addition and the importance of root morphological traits and N-use efficiency in affecting biomass. Therefore, these can provide important insights into potential changes of native species survival and development in nutrient-limited desert grassland caused by N deposition.

Native species are the main components of species composition and the basis of communities stability, its changes have been used to provide speci c signals from environmental change and make sensitive environmental monitors (Philippi et al. 1998). Thus, understanding the sensitivity of native species to nitrogen addition (e.g. N critical loads or saturation thresholds) is key to predicting ecological impacts of climate change in desert ecosystems. N saturation threshold, which have been widely considered with signals of ecosystem responses to N enrichment, is de ned as the point when ammonium and nitrate availability exceed plant and microbial nitrogen demand (Aber et al. 1989

Experimental conditions and treatments
The experiment was conducted from 02 July 2020 to 8 January 2021 in a climate-controlled greenhouse at the Agricultural Research Center of Ningxia University (38°30′11″N, 106°8′25″E), Yinchuan, Ningxia Province, China. The relative humidity in the glasshouse was about 60%, and day/night temperature was 28/15±2°C. The average light intensity ranged from 22.6 to 184.2mmol m −2 s −1 , and the photoperiod was set at 16 hours (natural light was supplemented with high-pressure sodium lamps on cloudy days, and early in the mornings and late in the evenings).
The seeds of native A. mongolicum keng were collected, originating from desert steppe within Sidunzi Village (37°20'30″N, 107°15'38″E) in Yanchi County in Ningxia, China, a location at which these species are commonly found. Ten healthy seeds of A. mongolicum were surface sterilized in 5% NaClO 3 for 10 min, rinsed three times in distilled water and then sown in plastic pots (25 cm top diameter×27 cm height) lled with 6 kg of sandy soil collected from the top 30 cm of soil from vacant land on desert steppe, where was similar to where collected seeds of A. mongolicum keng. The organic matter content of the untreated soil was 2.01 g kg −1 , the pH was 9.07, and the alkali-hydrolyzable nitrogen, available phosphorus and available potassium concentrations were 8.26mg kg −1 , 8.04mg kg −1 and 119.2mg kg −1 , respectively.
The experiment comprised ve N addition treatments (N0, ambient; N0.8, ambient + 0.8 g N m -1 yr -1 ; N1.6, ambient + 1.6 g N m -1 yr -1 ; N2.4, ambient + 2.4 g N m -1 yr -1 ; and N4, ambient + 4 g N m -1 yr -1 ) and six replicate pots. The low level of N addition at the N1.2 treatment (1.2 g N m -1 yr -1 , data not shown) was nearly equivalent to the background deposition rate (1.2 g N m -1 yr -1 ) in the study area (Bai et al. 2020). N additions were applied through Urea [CO(NH 2 ) 2 ] at twice equal applications, which dissolved in 300ml of tap water to add at the time of seeding and at the time of tillering on, respectively. The same amount of tap water was added to the N control pots during each fertilization event. All plants were irrigated every 2 days with plain tap water (300 500 mL per pot) and maintained soil water content at 70-80% of eld capacity during the experiment. All pots were randomly arranged in the greenhouse. All the plants were harvested for further analysis on January 8 th , (the ve weeks after the last N application).

plant and soil sampling
Whole tillers of individuals from the different treatments (six replicated pots) were dug up and separated into roots and shoots (leaves and stem). The roots were washed using deionized water, and the water on the surface of the roots was wiped with lab tissue paper. Then, all the harvest samples were divided into two subsamples. One subsample was packed into tinfoil and immediately immersed in liquid N 2 for transportation to the laboratory, and stored at −80°C for further physiological analysis. The other subsample was taken to the laboratory and stored at 4 °C for morphological properties analysis.
For each treatment, four of the six replicated pots were randomly selected for soil analysis after the plants harvested (n = 4). Soil samples were sieved through a 2-mm mesh to remove roots and placed in individual plastic bags and then immediately stored in a portable refrigerator to take the laboratory. To determine the response of soil N availability, including NH 4 + -N and NO 3 − -N, to N addition, 30 g of each soil composite sample was packed into a small plastic bag, and then immediately stored at 4 °C in the laboratory by using standard methods. The rest of each soil composite sample was air-dried for analyzing soil available phosphorus (AP).

Measurement of NUE traits and soil available nutrients
Dried samples of root and shoot biomass in each treatment were milled in a ball-mixer mill (MM200, Retsch, Haan, Germany) and then kept in a 1.5-ml vial at 60 °C to avoid water sorption. The samples 2.5 mg of pulverized material was placed in a tin capsule and then analyzed for total N concentration using elemental analyzer (Flash EA1112, Thermo Scienti c, West Palm Beach, USA). The total N accumulation was obtained as the product of total N concentrations and plant total dry weight (Iqbal et al. 2020b). NUtE was measured as total plant dry weight divided by N concentrations and NUpE was determined as

Measurements of biomass and root morphological traits
After the plants were harvested, ve plants per treatment from six replicated pots were randomly selected and separated into roots and shoots (leaves and stem), and then the root parts were carefully washed with tap water. Determination of total soluble protein, free amino acid, starch and total soluble sugar content Total soluble protein content(SPC) in roots and shoots was measured according to the supposed method by (Marion et al. 1976) using Coomassie Brilliant Blue (G-250) as a dye and albumin as a standard (Balasubramanian and Sadasivam 1987). For the determination of total free amino acid content (FAAC), the ninhydrin method was used as reported previously (Yokoyama et al. 2003) with some modi cations (Iqbal et al. 2020b). The total amounts of soluble sugar content(SSC) and starch content (SC) in tissues were determined using the anthrone method as described previously (He et al. 2013) .

Statistical analyses
Data were checked for normality and homogeneity of the residuals using Levene's Test prior to analyses.
Log transformation was applied when data did not meet the criteria for normality and homogeneity. One

Results
Effects of N addition on A. mongolicum Keng biomass Shoot biomass of A. mongolicum Keng were signi cantly increased with the increasing N addition. Compared to the control group (N0), shoot biomass was signi cantly improved by 100.51% under N1.6 and 219.76% under N2.4, respectively, with the highest value at N2.4 treatment (P <0.001; Fig. 1a). Although no signi cant effects of N addition on the root biomass of A. mongolicum Keng were observed, tending to increase from N0 to N2.4 but decrease from N2.4 to N4.0 (P = 0.058; Fig. 1a). Meanwhile, a quadratic equation can also be used to describe the relationship (R 2 = 0.657) between the total biomass and the N addition gradients exhibited a unimodal response with the increasing N addition gradient, and the N saturation threshold was around 3.2g N m -1 yr -1 (Fig. 1b).

N addition effects onsoil available nutrient properties and root morphological traits
We found that there were signi cant effects of N addition on soil NO 3 − -N concentration (P = 0.000; Table   1) and available phosphorus concentration (AP) (P = 0.000; Table 1), respectively. Both of them signi cantly increased with increasing N addition from N0 to N4 and showed linear response to N addition. While soil NH 4 + -N concentration showed negative response to N addition, the difference is not signi cant (P = 0.14; Table 1). Simultaneously, the root morphological traits response to N addition was observed (Table 1and Fig . 2). Root surface area (RSA) and volume (RV) showed extremely signi cant responses to N addition (both P = 0.000; Table 1), total root length (RL) and root forks number (RF) showed signi cant response to N addition (both P = 0.002; Table 1), as well as all of them tended to increase with increasing N addition rates from N0 to N2.4 but decreased above N2.4, with the higher value at N2.4 treatment (Table 1). However, N addition did not affect root diameter (RD) (P = 0.23; Table  1). In addition, linear regressions showed that root morphological traits were signi cantly and positively related to biomass of A. mongolicum Keng (all P < 0.01; Fig. 2).

Responses of activities of N metabolism enzymes and assimilation products in shoot and root to N addition
The activities of N metabolism enzymes in shoot and root were altered by increasing N addition (all P < 0.001; Fig. 3). N addition signi cantly improved the NR, NiR, GOGAT, GS and GDH activities in both the shoot and root (Fig. 3a, b, c, d and e). The N2.4 treatment signi cantly increased all the N metabolism enzymes activity compared with the N0 treatment, while there was not signi cantly changed when N addition treatment were more than N2.4 (Fig. 3).
Responses of N-use e ciency in A. mongolicum Keng to N addition N-use e ciency, like N uptake e ciency (NUpE) and N utilization e ciency (NUtE), was signi cantly affected by N addition. NUpE (F = 16.597, P = 0.000; Fig. 5a) and NUtE (F = 15.068, P = 0.000; Fig. 5a) signi cantly increased with increasing N addition rates from N0 to N2.4 but signi cantly decreased more than N2.4. We also estimated the relationships between biomass A. mongolicum Keng and NUE, and found biomass was increased linearly by NUpE and NUtE, respectively (Fig. 5b).

Direct and indirect effects soil available nutrient and N-use e ciency on biomass
Our structural equation models tted the data well, indicating that N-induced changes in soil available nutrient other than direct affect, but have an indirect impaction biomass of A. mongolicum Keng via regulation of root morphological traits and N-use e ciency (χ2 = 2.292, P = 0.994, GFI=0.969, RMSEA < 0.001; Fig. 6). This model showed that N addition directly explained 80.5% of variance in soil available nutrient (Fig. 6). In accordance with the results of ANOVA (Table 1), N addition had positive effects on soil NO 3 − -N and AP concentration (All P < 0.05; Table 1, Fig. 5). It also showed that pathways of soil available nutrient, root morphological characteristic, N metabolism enzymatic activity, N assimilation products and N-use e ciency together explained 71.7% of the total variance in biomass (Fig. 6). All of them had a strong and direct effect on biomass (Fig. 6). Additionally, soil available nutrient contributed weights of up to 16.2% and 84.4% to biomass via in uencing the root morphological characteristic and N-use e ciency, respectively (Fig. 6), indicating that soil available nutrient also had an indirect positive effect on biomass. However, N metabolism enzymatic activity displayed a signi cantly negative effect on biomass, but this effect became strong positive via the pathway of N-use e ciency (Fig. 6).

Nonlinear responses of A. mongolicum Keng to N addition
Many studies have con rmed that N addition has a positive effect on plant growth, but this effect will decrease and even diminish when the N inputs surpass the N saturation threshold (Tian et al. 2016;Zong et al. 2016). In this study, N addition promoted the root and shoot biomass of A. mongolicum Keng at low levels of N addition but decreased at high levels of N addition (Fig.1a), and total biomass exhibited a nonlinear response to the increasing N addition gradient, with a threshold at 3.2g N m -1 yr -1 (Fig. 1b), this result was in agreement with this study's initial hypothesis and consistent with previous studies.  ) also conducted a eld experiment in temperate steppe with six N rates, he found that grasses species richness showed a weak change across N treatments, but species richness of forbs declined more strongly. Furthermore, our results also showed that shoot biomass revealed more signi cant changes than root after N fertilization, indicating that shoot was more sensitive to N addition. As documented previously, most terrestrial ecosystems have received an accumulation of 2-5 g N m −2 ambient N deposition (Tian et al. 2016), which suggests that plant species in global terrestrial ecosystems likely to have the risk of N saturation and loss in the face of potential future climatic and environmental changes. Although the response of whole community cannot be evaluated by individual species in this study, our result that this N saturation threshold (3.2g N m -1 yr -1 ) in A. mongolicum Keng may provide speci c signals for restoring and managing the biodiversity and ecosystem functioning of desert steppe ecosystem in the future.

Analysis of mechanisms underlying for biomass nonlinear responses to Naddition
The soil on the desert grassland ecosystem in Yanchi are poor and N-limited (Gao et al. 2014), and external N inputs can adjust to this situation. Results from our study showed that N addition enhanced soil available nutrient (Table 1). Soil NO 3 − -N and AP showed a signi cant, linear increase with N addition (Fig.6) and no an appearance of threshold, consistent with that of a previous study (Bowman et al. 2006) which showed that plant responses rather than soil responses may provide more sensitive signal of the in uences of atmosphere N deposition. Simultaneously, results from SEM analyses showed that soil available nutrient generated a signi cant positive effect on biomass of A. mongolicum Keng (P < 0.001; Fig.6), indicating that response of A. mongolicum Keng to N addition may be partly and directly explained by soil available nutrient. Additionally, in our experiment, N:P ratio of soil available nutrient had a positive relationship with dry biomass of A. mongolicum Keng (Fig.S6a), implying that nonlinear responses of A. In our study, we also found that soil pH values were signi cantly decreased with N addition (Fig.S6b), suggesting that the response of A. mongolicum Keng to N addition may be associated with soil pH (Yuan et al. 2020). For nonlinear responses to N addition, the evidence are also usually observed in soil microbial biomass, microbial reallocation of C and some other soil biogeochemistry processes ( showed that the response of A. mongolicum Keng was partly induced by root morphological characteristics changes following N addition (Fig.6), which was con rmed by our linear regressions between biomass and root morphological traits (Fig. 2a, b, c, d). This means, like other plants, that A.
mongolicum Keng supplied with external N will tend to have a root system that is larger and more e cient in absorbing nutrients and subsequently promote its growth. The root growth may become limited by other resources (e.i., carbohydrates) and nally ceases when the nutrients in the soil exceed an optimal level (Dupas and Monteiro 2018; Xu et al. 2012). In addition, growth adaptation to N uctuations is paralleled by the adjustment of N metabolism (Luo et al. 2019). Therefore, determination only of root morphological characteristics does not contribute much information for assessment of plant response to N addition.
Given that plant growth was tightly linked NUE by biochemistry process, N-induced changes in NUE can be regarded as potential mechanisms interpretating the responses of plants to N addition. The NO 3 absorbed by the root system is converted to ammonium by NR and NiR, and then is assimilated to Gln and Glu via the activities of GS and GOGAT. Alterations in N metabolism-related enzyme activities under different N levels cause the discrepant absorption and formation of N-containing compounds , and nally affect NUE (Iqbal et al. 2020a). In the present study, the increase of NR, NiR, GS, GOGAT and GDH at low N addition rates and decrease at high N addition rates were observed in the roots and shoots of A. mongolicum Keng (Fig.3), similar response to increasing N addition had also been found in the N-containing compounds (Fig.4), indicating that response of A. mongolicum Keng to N addition may be related to changes in N metabolism and assimilation. This is further supported by both of them negative effect on biomass and this effect became somewhat positive via the pathway of NUE (Fig. 6). Furthermore, we also found that N addition altered NUE. Speci cally, N addition caused a signi cant decrease in NUpE and NUtE when N addition rates are more than N2.4 treatment (Fig.5a), and this phenomenon has been widely considered as one of the most important mechanisms underlying the effects of N deposition on plant productivity (Dupas and Monteiro 2018; Iqbal et al. 2020b; Yang et al. 2011a). In support of this view, results from the SEM analyses showed that N addition generated a signi cant indirect effect on biomass via the pathway of N-use e ciency (P < 0.01; Fig.6). Meanwhile, linear regressions also demonstrated there are well-correlated relationships between biomass and NUE (Fig.5b). Therefore, the lower NUE as evidenced by declines of N metabolism and assimilation at high N addition rates may explain the nonlinear response of A. mongolicum Keng as observed in the current study. In addition, physiological and morphological adaptation to environmental conditions also have been driven by changes in gene regulation (Iqbal et al. 2020a;Luo et al. 2015), hence, genome research is needed to test this response of A. mongolicum Keng to N addition in the next research.

Conclusion
Our results indicated that the growth responses of A. mongolicum Keng to increasing N addition rates appeared a unimodal-shaped response with a N saturation threshold at 3.2g N m − 1 yr − 1 . Besides, Ninduced changes in soil available nutrient other than direct affect, have an indirect impaction on biomass of A. mongolicum Keng via regulation of root morphological traits and N-use e ciency. All these can provide important insights into potential changes of native species survival and development in a nutrient-limited desert steppe ecosystem caused by atmospheric N deposition.

Declarations
Author's contribution A. Y., B. C., and D.M. designed the experiment. A.Y conducted the experiment and X.W and X.J. assisted the eld measurements. A.Y. analyzed the data and wrote the manuscript, and B.C revised the manuscript. All authors provided comments on the manuscript and the revisions and approved the nal version.
Biomass of A. mongolicum Keng in response to N addition treatments. Means ± SE (n=5). Different letters indicate signi cant difference at P < 0.05. The arrow indicates the N saturation threshold for biomass of A. mongolicum Keng.

Figure 2
Root phenotype of A. mongolicum Keng under various N addition gradients (left) and the relationship between these root morphological parameters and total biomass (a-d).
Page 19/21  N assimilation products in shoot and root of A. mongolicum Keng in response to N addition treatments.
Different letters above bars indicate signi cant differences among the N addition treatments (P < 0.05). The values are presented as mean ± SE (n = 4). Figure 5