N addition alters non-structural carbohydrates and C:N:P stoichiometry in different organs of Reaumuria soongorica seedlings in Gobi Desert of the Northwest, China

Reaumuria soongorica is an important biological barrier for ecological protection in Gobi desert of the Northwest, China, where soil nitrogen availability is low. N deposition increasing signi�cantly in Gobi desert, so that the response of R. soongorica to N enrichment may become a problem. However, little is known the effects of N addition on biomass, non-structural carbohydrates (NSC), and carbon:nitrogen:phosphorus (C:N:P) stoichiometry of R. soongorica in this region. Here, we examined changes in biomass, NSC and C:N:P ratios of different organs for one year growth of R. soongorica at four N treatments which is 0 (N 0 ), 4.6 (N 1 ), 9.2(N 2 ), and 13.8 (N 3 ) g m −2 year −1 . The N addition ( reach to 9.2 g m −2 year −1 ) signi�cantly enhanced the biomass of different organs, meanwhile the belowground:aboveground ratio was also signi�cantly increased. The NSC concentration of root signi�cantly enhanced under N addition treatments, but the stem and leaf NSC concentration was only increased signi�cantly at N 1 and N 2 addition. N addition only enhanced the soluble sugar concentration of leaf and root, and reduced starch concentration of different organs. The N concentration of stem and root were signi�cantly enhanced at N 2 and N 3 addition and the leaf N concentration was only increased in N 3 addition, but N addition had no signi�cant effect on C and P concentrations. The stem and leaf C:N ratio were reduced signi�cantly in N 2 and N 3 treatments, but the root C:N ratio was decreased signi�cantly in N addition. The N 3 addition signi�cantly increased N:P ratio of different organs. Our results suggested N addition changed the biomass, NSC, N concentration, C:N and N:P ratio of different organs, meanwhile the root responded more strongly than stem or leaf, this is bene�cial for absorbing more water in arid soil in this region, then to ensure the survival of R. soongorica seedlings.


Introduction
Since the industrial revolution, atmospheric nitrogen (N) deposition has increased rapidly due to the widespread use of N-containing synthetic fertilizers and increased fossil fuel combustion, and the in uence of N deposition on land ecosystems has become one of the research hotspots 1,2 . The study showed that N deposition affected plant photosynthesis and nutrient transport 3 , but as the main product of photosynthesis, the existence form and distribution of carbohydrates in plant organs will been affected by N deposition 4 . Meanwhile N deposition has also been shown to affect the stoichiometry(C, N, P) of plant in terrestrial ecosystems and thus alter the physiological activity and growth rates 5 .
The non-structural carbohydrate(NSC) include soluble sugars and starch, it is the main energy supply substrate for plant growth, development and reproduction, and it plays the function of metabolism, transporting water and assimilation products, osmotic regulation and reservoir pool 6,7 . So understanding the NSC concentration of plants and its changes in various organs is the ideal entry point to explore the response and adaptation of plant growth and physiological ecological process. Facing environmental stress, plants will change C allocation among different organs. For example, drought either signi cantly increased or maintained the total NSC concentration in the above-ground organs, but reduced the total NSC concentration in the sapling roots 8 . Compared to without any fertilization, the NSC in leaf, stem, and root of Moringa oleifera seedlings were greatly reduced by N and P fertilization which could be explained by the dilution effects of increased biomass following fertilization 9 . It is seen that NSC and its components (soluble sugar and starch) showed different responses to different environmental factors among different organs 10,11 . So understanding the change pattern of NSC under different environmental stress, has become an important means to explore the plant adaptation strategy under different environmental stress.
Carbon (C), nitrogen (N) and phosphorus (P) are structural elements and the major nutrient elements that play an important role in maintaining the biogeochemical cycle and ensuring both nutrient cycle and energy ow within ecosystem 12 . Many researches found N addition signi cantly increased the C, N and P contents of plant organs 13,14,15 . Plant C:N and C:P ratios usually increased 16 or decreased 17 under N addition, but plant N:P ratio maybe increased 15 , decreased 18 or maybe not affected 17 depending on species 18 and soil nutrient conditions 19 . However, most studies have focused on aboveground stoichiometries rather than the level of whole plants 20,21 , the responses of plant different organs to N addition remain largely unexplored in Gobi desert region.
Under natural conditions, R. soongorica can reproduced by seeds, The success of its natural renewal mainly depends on the seed germination and seedling survival conditions. For Gobi desert regions, nitrogen was a key factor which restricted vegetation growth, distribution and the restoration of damaged ecosystems, so the germination of R. soongorica seeds and the survival of seedlings are bound to be affected by nitrogen. N deposition maybe is bene cial for the R. soongorica growth, but the researches about R. soongorica had focused on growth 22,23 , photosynthetic physiological properties 24,25 and genes 26 in recent years, however little is known about the biomass, C:N:P stoichiometry and NSC distribution in R. soongorica seedlings under N addition in Gobi desert region of the northwest China, especially the differences among different organs. So in order to evaluate the distribution of C:N:P stoichiometry and NSC content for different organs and how nitrogen changes may alter these distribution for R. soongorica, we hypothesized that (1) N addition would signi cantly enhance the different organs biomass, but the aboveground (stem and leaf) biomass would respond more strongly

Biomass in different organs
With increasing N addition, the total, stem and root biomass was increased signi cantly and reached the maximum value at N 2 addition treatment (P<0.05). Compared with N 0 treatment, the total, stem, leaf and root biomass increased by 60.49%, 48.86%, 42.85% and 97.5% in N 2 addition treatment, respectively. The ratio of belowground to aboveground biomass was also signi cantly increased in N 1 and N 2 treatment because the increase of root biomass was more rapid than that of stem and leaf biomass (P<0.05), but then reduced in N 3 addition treatment (Table1).

NSC concentration in different organs
The soluble sugar concentration, starch concentration and NSC concentration for different organs was different with increasing N addition. With increasing N addition, the soluble sugar concentration for stem, leaf and root were all increased signi cantly at the N 1 and N 2 addition treatments, but then it decreased signi cantly at the highest N 3 treatment except of the stem (

Nutrient concentrations and stoichiometry in different organs
The N addition had no signi cant effect on concentration and P concentration of different organs ( Fig. 2a, 2c). Compared with N 0 , The N concentration of stem and root signi cantly increased in N 2 and N 3 addition treatments; the leaf N concentration signi cantly enhanced in N 3 addition treatment (P<0.05).
The N concentration of different organs had no signi cant difference between N 0 and N 1 addition treatment (Fig. 2b).
The stem and leaf C:N ratio was all reduced signi cantly in N 2 and N 3 addition treatments, but the root C:N ratio was decreased signi cantly with increasing N addition(P<0.05) (Fig. 2d). Compared with N 0 , the C:P ratio of different organs all had some decreasing, but it had no signi cant difference among N addition treatments (P>0.05) (Fig. 2e). The higher N addition (N 2 -N 3 ) signi cantly enhanced the stem N:P ratio, but the leaf and root N:P ratio was only enhanced in N 3 addition treatment (Fig. 2f) (P<0.05) .

Discussion
Effect of N addition on plant biomass. Plant growth is closely related to nutrients, especially for nutrientde cient soil. Soil nitrogen content is lower and it become one of the main limiting factors for plant productivity in arid and semi-arid region of northwest China 27 . Contrary to our rst hypothesized, we found N addition( reach to 9.2g m − 2 year − 1 ) had signi cantly enhanced the biomass of different organs, but it was reduced signi cantly at higher level(13.8 g m − 2 year − 1 ), this is consistent with the results of Jin et al. 13 this nding indicates that appropriate N addition is bene cial to the growth of R. soongorica in Gobi desert region, but if the N addition exceeded the minimum N demanded of plant growth, the plants can be less sensitive to N addition 28 .
The previous study showed that plants can increase their allocation of photosynthetic products to belowground organs under lower 0.15mM N condition 29 , but with the N addition, the biomass allocation are species-speci c 4,30 . We found the response of roots biomass to N addition( reach to 9.2g m − 2 year − 1 ) was signi cantly higher than the shoot biomass, thus result in higher belowground:aboveground ratio, this was inconsistent with the previous study 4 and our hypothesis. The reason maybe is in order to absorbed more water from the soil in Gobi desert region, the root biomass of R. soongorica was increased more than the stem and leaf under N addition condition.
Effect of N addition on NSC. NSC is the product of photosynthesis and provide energy for plant growth and metabolism, meanwhile it play an important role in the response of plants to environmental changes 31 . Some studies reported that N addition increased the NSC accumulation 30 , but other studies found N addition had no effect on or even decreased NSC concentration 4,9 . In this study, we found a signi cantly increase in NSC concentration of different organs of R. soongorica within a certain range of N addition (0 -9.2 g N m − 2 year − 1 ), but it had some decreasing in highest N addition (13.8g N m − 2 year − 1 ), this mainly due to signi cant increasing in soluble sugar concentration and reducing in starch concentration, but this nding not supported our third hypothesis and was consistent with the results of other studies 4,32 . Meanwhile some research reported the accumulation of higher NSC concentrations can balance the osmotic pressure of cells 33 and be used to cope with environmental stress, thus the highest NSC concentration in different organs under middle N addition (9.2g m − 2 year − 1 ) in our study indicated adding proper amount of N will bene t for the NSC accumulation and improve the resistance of R. soongorica to water stress in Gobi desert region, and then it is bene cial for the successful settlement of R. soongorica in this region .
In this study, signi cant NSC increasing was observed in roots under N addition condition than stem and leaf NSC, this is consistent with other research 4,30 , but the difference is the root NSC was higher than stem and leaf NSC in our study, Ai et al 4 found the aboveground NSC was higher than belowground NSC, this difference may be related to the characteristics of carbon allocation and transport 34 . In order to facilitate water absorption and survive in Gobi desert region, the more carbon of R. soongorica allocated to the root than stem and leaf 35 .
Effect of N addition on plant stoichiometry. Stoichiometry re ected the utilization ability of C, N and P for plant, which are susceptible to environmental changes 36 . Many studies have concluded that N addition will resulted in higher soil N availability, and thus enhanced N concentrations and declined C:N ratios in many species 37,38 , some researches also found N addition signi cantly increased the C and N contents of plants and no signi cantly effect on C:N ratio 14,39 . In this study, we found that N addition increased the N concentration and reduced C:N ratio of R. soongorica, but no signi cantly effect on N and P concentrations and C:P ratio, this is consistent with our second hypothesis and many previous studies 17,40 . N:P ratio has been interpreted as an indicator of N and/or P limitation 41,42 . It is widely accepted that N:P ratios < 10 indicate N limitation. Therefore, R. soongorica shows very low N:P ratios due to relatively high P concentrations in higher N addition treatment, it is indicated this plant would still be N limited despite the massive N doses that were applied. this is consistent with the result of Wang et al. 43 who found the N:P ratio of R. soongorica was lower in the arid desert region, but it is inconsistent with the result of Niu et al. 44 who found the N:P ratios of six shrubs(including R. soongorica) in the desert of the Alxa Plateau all were higher. This inconsistent results maybe related with ecosystem types and environmental factors 45 .
The responses of stoichiometry of different organs was different when environmental changes 46 . For example, some studies showed that leaf N and P concentration and N:P were higher than those of the other organs under N addition 15 . Xiao et al. 30 reported that N addition increased N concentration in roots than shoots, but decreased P concentration and increased N:P in all organs. In this study, we found N addition signi cantly increased N concentrations and decreased C:N the in roots than aboveground organs, this supporting our second hypothesis, the reason maybe is that N addition enhanced soil N availability, but the absorbed N by roots can not be transferred to the aboveground organs because of lower soil water in Gobi desert region.

Conclusions
The total and other organs biomass signi cantly enhanced with a range of N addition(0-9.2g m − 2 year − 1 ), and the increasing of root biomass more rapidly than the stem and leaf biomass which resulted in higher belowground:aboveground ratio. N addition enhanced the soluble sugar concentration, but reduced the starch concentration of all organs, and the NSC concentration of root signi cantly enhanced at all N addition, but the stem and leaf NSC concentration was only increased signi cantly at N 1 and N 2 addition. The N concentration was increased at higher N addition(9.2-13.8g m − 2 ), but it had no signi cant effect on C and P concentrations, thus resulted in the C:N ratio reducing signi cantly. This indicated N addition changes the biomass, NSC, N concentration, C:N and N:P ratio of different organs, meanwhile the root responded more strongly than stem or leaf.

Materials And Methods
Plant materials and growth conditions. Seeds of R. soongorica were harvested in October 2017 from the Gobi experiment elds at the Linze Inland River Basin Research Station (39°21′ N, 100°02′ E, 1400 m asl), Chinese Academy of Sciences, the seeds were saved in the sealed plastic bags at a laboratory.
The experiment was also conducted at the Linze Inland River Basin Research Station. The average annual precipitation of 117 mm and average temperature is 7.6°C. The brown desert soil was used in our experiment which was collected from the upper 20 cm layer of Gobi experiment eld in October 2017, the organic matter concentration of soil is 3.9 g kg -1 and the PH is 9.0, the available N and P is 0.19 and 0.21 g kg -1 , respectively. The soil was sieved through a 5 mm mesh before the experiment. Experimental design. In the March 2018, the soil was added to pots with an diameter of 34 cm and a height of 40 cm, then the seeds of R. soongorica were sown into the pots, the soil water content was maintained at the above 80% of eld capacity during the seedling establishing. After emergence, two seedlings of R. soongorica were retained in each pot for one year growth. The experiment was started in May 2019 and the excess plant was removed to maintain one seedling in each pot.
The nitrogen level was designed according to the nitrogen deposition level in the desert region 47 , four treatments received additional N in the form of urea at the rates of 0 (N 0 ), 4.6 (N 1 ), 9.2(N 2 ), and 13.8 (N 3 ) g N m −2 year −1 . Each treatment had six replicates. The urea was selected as N source and the N content is 46.7% , so the amount of urea was 0.8865, 1.7730, 2.6595g for N 1 , N 2 , N 3 treatments. The N was applied in the beginning of May, June, July, August, September and October in 2019, as a solution of urea in the same volume deionised water, and the N 0 received the same volume of water in the same time.
Sampling and chemical analysis. The six seedlings of R. soongorica for each treatment were sampled in October. The whole seedlings of R. soongorica were taken out from the pots when sampling, then the roots were wrapped with plastic wrap to prevent water loss, and all samples were taken back to the laboratory. The seedlings were divided into 3 parts: leaves, stems and roots, then every part was washed by distilled water and excess water in the surface was removed by blotting paper. All samples were weighed, then they were placed in labeled envelopes at 120℃ for 30 min in an oven and oven-dried at 80℃ for 48 h to assess the dry mass. At last, the dried samples ground through a 40-mesh sieve and stored for chemical analysis.
The NSC concentration was calculated as the soluble sugar concentrations plus starch concentrations in this study 4 and the concentrations of the soluble sugars and starch were determined using an anthrone method 48 . Total N concentrations were determined using the Kjeldahl method. Total P concentrations were determined using the molybdenum blue colorimetric method. Total C concentrations were determined using the dichromate oxidation method 49 .
Data analysis. A one-way analysis of variance (ANOVA) to test the effects nitrogen on biomass, the concentrations and stoichiometries of NSC, C, N and P of different organs. Duncan's multiple range tests was used for multiple comparisons among each treatment. Data analyses were performed using SPSS Statistics procedure (version 13.0) and gures were drawn by Origin 8. Declarations construction fund project of Gansu Agricultural University (GAU-XKJS-2018-104, GAU-XKJS-2018-108). We thank Linze Inland River Basin Research Station, Chinese Academy of Sciences, for providing the experiment site.
Author contributions TTX analyzed the data and wrote the manuscript. WTZ conducted the experiment and performed the measurements. SLS participated in the planning of the study, data analysis, and writing of the manuscript.

Competing interests
The authors declare no competing interests.

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Figure 2
Effects of N addition on plant biomass C, N, and P concentrations and their stoichiometric ratios of C:N, C:P, and N:P of R. soongorica. Error bars are SE (n = 6). Different lowercase letters above bars indicate signi cant difference at P = 0.05

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