Global Ecology and Conservation

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Original Research Article

Bene ﬁ cial effects of nitrogen deposition on carbon and

nitrogen accumulation in grasses over other species in Inner Mongolian grasslands

Xue Wang

^a^,^b^,^*

, Meng Wang

^a

, Yimin Tao

^a

, Nina Fang

^a

, Guojiao Yang

^b

, Jiangping Cai

^b

, Yong Jiang

^b

, Xingguo Han

^b

, Fei-Hai Yu

^a

, Mai-He Li

^b^,^c^,^d

aInstitute of Wetland Ecology&Clone Ecology, Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation, Taizhou University, Taizhou, 318000, China

bErguna Forest-Steppe Ecotone Research Station, CAS Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, 110016, China

cForest Dynamics, Swiss Federal Research Institute WSL, Zuercherstrasse 111, CH, 8903, Birmensdorf, Switzerland

dKey Laboratory of Geographical Processes and Ecological Security in Changbai Mountains, Ministry of Education, School of Geographical Sciences, Northeast Normal University, Changchun, 130024, China

a r t i c l e i n f o

Article history:

Received 27 October 2020

Received in revised form 14 February 2021 Accepted 15 February 2021

Keywords:

Forb Grass Legume Nitrogen addition Non-structural carbohydrate Starch

a b s t r a c t

Globally, chronic nitrogen (N) deposition into terrestrial ecosystems has resulted in changes in community composition, depending on the responses of co-existing species to increasing soil N availability. The levels of plant non-structural carbohydrates (NSCs) produced by photosynthesis are associated with leaf N content, and together they represent the capital of a plant for its life including competitive ability. However, the speciﬁc ways that concentrations of plant NSCs, and also N, inﬂuence the growth of plants, and thus their productivity, are still abscue. Here, we explored the effect of variations in plant leaf NSCs (starch and soluble sugars) and N concentrations on the growth of plants with different soil N uptake capacities, i.e. two grasses (Leymus chinensisandStipa baicalensis), two forbs (Potentilla tanacetifoliaandGalium verum), and one legume (Thermopsis lanceolate) in Inner Mongolian grasslands subjected to three N addition rates (i.e., 0, 10, or 20 g N m²yr¹) and three N forms [i.e., NH4NO3, (NH4)2SO4, or CO(NH2)2] for four years.

Irrespective of N addition rate and N form, N addition signiﬁcantly increased aboveground biomass of the grasses, but not that of the forbs or the legume. The grasses had higher increase in N concentrations than the forbs and the legume. N addition increased the starch concentrations of grasses, but decreased that of forbs and the legume. Changes in the aboveground biomass of all tested plant species were signiﬁcantly positively correlated with changes in concentrations of both NSCs and N. Our results indicate that, irrespective of the N addition rates and N forms, greater increase in NSCs and N concentrations contributed to higher aboveground productivity in grasses, but not in forbs and legumes.

These results imply that future nitrogen deposition will beneﬁt grasses over other plant forms, and thus the former may become more dominant in grasslands in northern China.

*Corresponding author. Institute of Wetland Ecology&Clone Ecology; Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conser- vation, Taizhou University, Taizhou, 318000, China.

E-mail address:wangxueiae@163.com(X. Wang).

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j o u r n a l h o m e p a g e :h t t p : / / w w w . e l s e v i e r. c o m / l o c a t e / g e c c o

https://doi.org/10.1016/j.gecco.2021.e01507

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1. Introduction

The amount of nitrogen (N) deposited into terrestrial ecosystems has signiﬁcantly increased due to an upsurge in anthropogenic activities (Erisman et al., 2013;Liu et al., 2013). This N increment can differentially affect the growth and competitive capacity of different plant species and functional groups in plant communities, resulting in a shift in the dominance of different species and functional groups (Cleland and Harpole, 2010). For instance, simulated N deposition has generally been shown to increase the abundance of grasses and decrease that of forbs in grassland communities (Xia and Wan, 2008;Xu et al., 2015;Fu and Shen, 2016;Mitchell et al., 2017;Tang et al., 2017).

The growth-related bio-physicochemical processes of a plant, such as carbon balance and N uptake, could signiﬁcantly respond to N addition (Pornon et al., 2019;Li et al., 2020). For example, the level of non-structural carbohydrates (NSCs, i.e.

soluble sugars and starch) in plant tissues is the result of carbon gain (photosynthesis) minus consumption (growth and maintenance respiration) (Chapin et al., 1990;Li et al., 2013), representing a plant’s capital for growth and defence (Li et al., 2002,2018). The photosynthetic capacity and thus the amount of NSCs produced by photosynthesis are positively correlated with leaf N (Evans, 1989) and soil N availability (Zhao et al., 2005). Therefore, N supply influences plant photosynthesis, NSCs status and growth, and further affects their productivity and ultimately their survival in a plant community. However, the specific ways that concentrations of plant NSCs and N influence the growth of plant species with different competitive N uptake capacities, such as grasses vs. forbs/legumes, are still obscure.

Plant species vary greatly in their N uptake capacities, depending on their physiological and biochemical characteristics and different soil N forms (Song et al., 2012;Sheppard et al., 2014;Yan et al., 2019), and thus show different magnitudes and directions of plant N responses to N addition with various soil N forms (Liu et al., 2016;Du et al., 2020). Consequently, plants exhibit differential responses of NSCs that are closely associated with N addition rates, N application duration, and even the specific N compounds applied (Humbert et al., 2016;van den Berg et al., 2016). For instance, some plants have higher NO3but lower organic N absorption capacity than others, even under optimal N environments (Nordin et al., 2006). Plants that dominate acidic soils absorb more NH4þrelative to other plants (van den Berg et al., 2008). Thus, the bioavailability of soil N in the various forms affects plant leaf N levels, which in turn influences photosynthesis and NSC concentrations, andfinally plant growth. However, we still lack information on how different soil N forms affect the relationship between carbon balance and the growth of different species in grassland ecosystems.

Increased aboveground biomass and cover of grasses, accompanied with decreased biomass and cover of forbs, were widely reported in N addition experiments in Inner Mongolian grasslands (Song et al., 2011,2012;Xu et al., 2012,2015;Zhang et al., 2015). To our knowledge, however, the plant carbon- and nitrogen-physiological mechanisms underlying those changes have rarely been studied. Thus, we examined the relationships between aboveground biomass and the concentrations of leaf N and NSCs (i.e. soluble sugars and starch) in two C₃grasses (Leymus chinensisandStipa baicalensis), two forbs (Potentilla tanacetifoliaandGalium verum), and one legume (Thermopsis lanceolate) in an Inner Mongolian grassland ecosystem subjected to various N addition rates and N forms for four years. We tested the following hypotheses: (1) N addition leads to a higher accumulation of leaf N and NSCs in grasses than in forbs and legumes, and thus a higher productivity; and (2) the magnitude of the stimulating effects varies with N forms and N addition rates.

2. Materials and methods

2.1. Study site and experimental design

The experiment was conducted at the Erguna Forest-Steppe Ecotone Research Station (N5010⁰46.1⁰⁰, E11922⁰56.4⁰⁰), northeastern China. Theﬁeld experiment was carried out in a natural steppe that has remained fenced since 2013 to prevent livestock grazing. Before then, the grassland was annually mown for hay-harvesting. The site has a long-term mean annual precipitation of 363 mm and a mean annual temperature of 2.45 C (1957e2016). According to the US soil taxonomy classiﬁcation, the site has chernozem soil. The pH of the topsoil (0e10 cm) is 6.8e7.0, and the dominant species in the ecosystem areLeymus chinensis,Stipa baicalensis,Cleistogenes squarrosa,Thermopsis lanceolata,Cymbaria dahurica, andCarex duriuscula.

The study was part of a long-term, large experiment (Yang et al., 2019) with 60 treatments and eight replications. Our sampling involved only seven of the treatments, i.e. two rates of N addition (i.e., 10, and 20 g N m²yr¹) crossed with three forms of N compounds [i.e., NH4NO3, (NH4)2SO4, and CO(NH2)2], and a control (no N addition). The N addition experiment began in late May of 2014 and the treatments were applied in a completely randomized block design (eight blocks) in plots measuring 10 m10 m. In the plots with N addition, a certain amount of the N compounds were broadcasted by hand once a year. To avoid potentially confounding effects, similar quantities of sand (0.5 kg per plot) were applied in the control plots.

2.2. Sampling

In mid-August 2017, after four years of continuous N addition, leaf samples were collected from six dominant or abundant species in each plot, including two grasses (Leymus chinensisandStipa baicalensis), two forbs (Potentilla tanacetifoliaand Galium verum), and two legumes (Thermopsis lanceolateandVicia sepium). The climatic condition during the sampling was shown inFig. S1. For each species, two to three healthy, fully expanded leaves were collected from each of 10 randomly

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selected plant individuals, if they were present, at the center of each plot to avoid edge effects. The samples were pooled to obtain a mixed sample for each species in each plot. We stored the samples in boxes with ice until they were oven-dried at 65C for 48 h and thenﬁnely ground to allow passage through a 0.15 mm mesh size. Since we could notﬁnd all the six species in all eight replicate plots, we used the four replicate plots where all the six species were found. However, the quantities of the samples ofV. sepiumobtained still did not meet the minimum requirement for analysis. Therefore we excluded this species from the analysis.

To assess aboveground biomass, we clipped all vascular plants at the soil surface in 1 m1 m quadrats (in mid-August 2017). The quadrats were randomly placed in each plot without a spatial overlap of the quadrats and at a minimum of 50 cm from the border of each plot to avoid edge effects. All living vascular plants were sorted according to species. The plant materials were then oven-dried at 65C for 48 h and then weighed.

Aﬁve mixed soil sample (0e15 cm soil depth) was taken from each plot, using soil cores with an inner diameter of 3.0 cm.

Fresh soil samples were sieved through a 2 mm mesh size, air-dried and ground intoﬁne powder before measuring total soil N.

Analysis of the concentrations of non-structural carbohydrates, nitrogen and chlorophyll content.

The powdered leaf material (0.1 g) was put into a 10 ml centrifuge tube, and 5 ml of 80% ethanol was added. The mixture was incubated in a water bath-shaker maintained at 80C for 30 min, then centrifuged at 3500 rpm for 10 min. The pellets were extracted at least twice with 80% ethanol. Supernatants were retained, pooled, and stored at 4C until further use for assessment of the total soluble sugars.

The ethanol-insoluble pellet was air-dried to evaporate the ethanol, and then the residue was used for starch extraction. To obtain the starch, the residue was dispersed in 2 ml distilled water and heated for 15 min in a boiling water-bath. After cooling to room temperature, 2 ml of 9.2 M perchloric acid (HClO4) was added, and the mixture was maintained at room temperature for 15 min to allow for starch hydrolysis. Subsequently, 4 ml of distilled water was added to each sample, and then centrifuged at 4000 rpm for 10 min. The pellets were used for starch extraction one more time with 2 ml of 4.6 M HClO₄. Supernatants were retained, combined, and topped up to 25 ml, and then used to determine starch concentrations. The concentrations of soluble sugars and starch were evaluated spectrophotometrically (ultravioletevisible spectrophotometer 752S, Cany Preci- sion Instruments Co., Ltd., Shanghai, China) at 620 nm by the anthrone method. Starch quantities were calculated by multiplying the glucose concentrations by a conversion factor of 0.9. Glucose was used as an internal standard (Li et al., 2008).

The chlorophyll content was determined colorimetrically by chloroplast pigment extract from 95% ethanol (Lichtenthaler 1987) (Fig. S2). A C/N analyzer (Vario Micro cube, Germany) was used to evaluate the concentration of leaf N and total soil N (Fig. S3).

2.3. Data analysis

For each of theﬁve species, we calculated the change (x) in each variable (aboveground biomass, N, soluble sugars, starch, and total NSCs) between each of the six N rate and N form treatments (T) and the control (C) as:

x¼variable in Tvariable in C variable in C 100%

A three-way analysis of variance (ANOVAs) was performed to determine the effects of plant species, soil N rate, soil N form, and their interactions on the change in aboveground biomass, N, soluble sugars, starch, and total NSCs in leaves (Table S1). We further used two-way ANOVAs to test these effects for each species separately. We also employed one-way ANOVAs to test the effect of species on aboveground biomass, N, sugars, starch, and NSCs in the control, and one sample t-tests to determine whether the change in each variable for each N rate and N form treatment (i.e. the difference between each of the six N rate and N form from treatments and the control) differed signiﬁcantly from zero. Tukey’s HSD was used for multiple comparisons.

Linear regressions were used to examine the relationships between the change in aboveground biomass and the change in N, soluble sugars, starch and total NSCs. All data were checked for normality with the Kolmogorov-Smirnov test and for ho- mogeneity of variance with Levene’s test. Data that did not meet the condition were transformed to log or square-root transformed prior to analysis. All statistical analyses were performed using the SPSS software version 22.0 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. Changes in aboveground biomass

The net changes in aboveground biomass varied among the different species (P< 0.001;Table S1). Compared to the control, N addition signiﬁcantly increased aboveground biomass ofL. chinensis(Fig. 1A,P<0.001) but decreased that ofP.

tanacetifolia (Fig. 1D,P¼0.017) and did not affect that of G. verumorT. lanceolate(Fig. 1C, E,P¼0.235 andP¼0.611, respectively). Neither two high N addition rates nor N forms affected the change in aboveground biomass of any of theﬁve species, except for a signiﬁcant effect of N rate forS. baicalensis, which had higher values with 10 g N m²yr¹(N10) than with 20 g N m²yr¹(N20) (Fig. 1B).

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3.2. Changes in leaf N and NSCs

In the control plots, the concentrations of N and NSCs in leaves varied among species (Fig. 2). The legumeT. lanceolatehad the highest leaf N concentration, and forbs had higher leaf N concentration than grasses (Fig. 2A). The concentrations of soluble sugars, starch, and total NSCs were in general higher in the forbs than in the grasses and the legume (Fig. 2BeD).

In the plots with N addition, the changes in the concentrations of N, soluble sugars, starch, and total NSCs in leaves varied significantly among species (P<0.001;Table S1). For all thefive species, we detected significant increases in leaf N concentration after N addition (Fig. 3AeE). However, the extent of the increase in the N concentration varied among species, and across the N rates and N forms, the grasses (þ44.4%) showed larger increases than the forbs (þ24.3%) and the legume (þ3.7%) (Fig. 3AeE,P<0.001). Neither two high N rates nor N forms affected the leaf N concentrations of any of thefive species, except forP. tanacetifoliawhere the N concentration was greater with the higher N addition rate (Fig. 3D). For L.chinensis,urea addition resulted in larger increases in the concentrations of soluble sugars and total NSCs than with the addition of ammonium nitrate (NH₄NO₃) or ammonium sulfate (NH₄)₂SO₄) (Fig. 3F, P,P¼0.037 for soluble sugars andP¼0.025 for total NSCs). Compared with the control, N addition led to an increase in the concentration of starch in the grasses (Fig. 3K and L), but a decrease in the forbs (Fig. 3M, N). Compared with the control, N addition significantly increased the sugar: starch ratio of the forbP. tanacetifolia(Fig. 3X).

3.3. Correlations between aboveground biomass and N, soluble sugars, starch, and total NSCs

Changes in aboveground biomass were positively correlated with the changes in the concentrations of leaf N, starch, and total NSCs (Fig. 4A, B, and D). However, there was no signiﬁcant correlation between mean changes in aboveground biomass and soluble sugars (Fig. 4C).

4. Discussion 4.1. Effects of N addition

The concentration of non-structural carbohydrates (NSCs) and starch in the grasses increased, whereas it decreased or unchanged in the forbs and the legume under N addition (Fig. 3KeO). Meanwhile, the grasses had a greater increase in leaf N concentrations than the forbs and the legume in response to N addition (Fig. 3AeE). Further, the changes in aboveground

Fig. 1.Changes in aboveground biomass of each plant species under six nitrogen (N) rate and N form treatments relative to the control. U, AN and AS stand for the addition of urea, ammonium nitrate, and ammonium sulfate, respectively; Nf and Nr represent for N form and N rate treatments, respectively.Pvalues of two- way ANOVAs are given. An asterisk above a bar indicates a signiﬁcant difference between the N addition treatment and the control (*P<0.05).

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biomass were positively correlated with the changes in leaf NSCs and N (Fig. 4). These results are consistent with ourﬁrst hypothesis that higher competitiveness regarding NSCs accumulation and N uptake contribute to higher productivity in grasses under N addition.

Exogenous N addition significantly increased aboveground biomass of the grasses and decreased that of forbs. Thisfinding is in agreement with previous studies (Bobbink et al., 2010;Tian et al., 2016;Mitchell et al., 2017;You et al., 2017). In our study, in response to N addition, the grasses exhibited a larger increase in leaf N concentrations than the forbs, as well as a greater increase in aboveground biomass. This increase in leaf N concentrations of grasses, according toYou et al. (2017), can be attributed to the branchedfibrous root systems in grasses which have the advantage of absorbing more nutrients from the soil than other species. Indeed, we found that rhizomatous grass (L. chinensis) and bunchgrass (S. baicalensis) had lower leaf N concentrations than the forbs and the legume in the control plots, but a larger increase in leaf N concentrations under N addition (Figs. 2A, 3AeE).

Exogenous N addition can enhance the photosynthetic rate of plants (Zhang et al., 2018;Liang et al., 2020) as well as leaf area development (Belanger et al., 1992), thereby increasing the concentrations of non-structural carbohydrates and subsequently increasing aboveground biomass (Niu et al., 2010). N addition resulted in an increase in both aboveground biomass and starch concentration in the grasses (Fig. 1A and B;Fig. 3K and L), probably because grasses have higher photosynthetic rates than forbs (Tjoelker et al. (2005), as we found a higher chlorophyll content in grasses than in forbs (Fig. S2). In general, the mobile carbohydates can be stored as starch in case that the carbon supply is greater than the carbon need. Thus, the strong competitive advantage in photosynthesis (starch accumulation) and the greater increase in leaf N concentrations in response to N addition contributed to the increase in aboveground biomass of grasses, especially forL. chinensis.

4.2. Effects of N addition rates

Although the higher N addition (20 g N m²yr¹) corresponded to higher total soil N concentrations (Fig. S3), it did not result in a significantly greater increase in leaf concentration (except for P. tanacetifolia), aboveground biomass or NSC concentrations in comparison to lower N addition (10 g N m²yr¹). According toAber et al. (1998), nitrogen levels exceeding a specific range could have an inhibitory effect on plants. Our results indicated that the biomass of the selected species could be saturated at a rate of>10 g N m²yr¹. This is in agreement withBai et al. (2010), who found that the aboveground biomass in Inner Mongolian grassland saturated at an N addition rate of approximately 10.5 g N m²yr¹. The legumeT. lanceolatewas less sensitive to N addition, which indicates that a surge in the amount of available N in the environment causes legumes to lose their advantage offixing N (Suding et al., 2005). Increased leaf N concentration can facilitate photosynthesis and promote the accumulation of mobile carbohydrates. On the other hand, N addition can accelerate growth, respiration, and other physiological processes that consume mobile carbohydrates (Hoch et al., 2003;Wang et al., 2017;Li et al., 2020). Thus, these processes could increase, reduce, or not affect the levels of mobile carbohydrates, depending on the dominant processes. In our study, For all the species in our study, no difference in mobile carbohydrate concentrations was detected between N Fig. 2.Concentrations of nitrogen and mobile carbohydrates (soluble sugars, starch, and NSCs) in each of thefive species in the control plots (without N addition).

Different lowercase letters indicate signiﬁcant differences among the different species (P<0.05).

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addition rates of 10 g N m²yr¹and 20 g N m² yr¹(Fig. 3). This result suggests that increased leaf N concentrations promote other processes, but not mobile carbohydrate accumulation.

4.3. Effects of N forms

Different soil N forms applied signiﬁcantly affected the concentration of mobile carbohydrates in grasses, although this effect did not induce any changes in aboveground biomass. This result is inconsistent with our second hypothesis.

Fig. 3.Changes in nitrogen (N), soluble sugars, starch, and total non-structural carbohydrate concentrations and in the sugar: starch ratio of each plant species between each of the six N rate and N form treatments and the control. U, AN and AS stand for the addition of urea, ammonium nitrate and ammonium sulfate, respectively; Nf and Nr represent N form and N rate treatments, respectively.Pvalues of two-way ANOVAs are given. An asterisk above a bar indicates the signiﬁcant a difference between the N addition treatment and the control (P<0.05).

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Across the species, aboveground biomass and leaf N concentrations did not vary among the different N forms, suggesting that thefive species did not exhibit differences in N uptake according to the form of N applied. The ratio of N to P is regarded as an important index to explain nutrient limitation pattern (He et al., 2006). According toKoerselman and Meuleman (1996) andGüsewell (2004), an N: P ratio<14 could imply N limitation. One possible explanation is that the limited soil N availability in this area (the N: P ratio is approximately 10 that is much less than 14) could facilitate an indiscriminate absorption of different N compounds. In our study, the mobile carbohydrates of the two grasses were more sensitive to different N forms than the forbs or the legume (Fig. 3). Mobile carbohydrates can be temporarily stored as starch for later use if the carbon supply exceeds the carbon demand of the plants. Under the additions of NH₄NO₃and (NH₄)₂SO₄, the two grasses had a lower accumulation of soluble sugars but higher accumulation of starch, suggesting a higher use efficiency of mobile carbohydrates in comparison to values at the treatment with urea. However, the changes in mobile carbohydrates induced by different N forms did not affect the aboveground biomass of grasses, indicating that mobile carbohydrates were sufficient (starch accumulation) for growth of the grasses, regardless of the N form applied.

Overall, our results showed that responses of leaf N and NSCs as well as the aboveground biomass to N addition differed signiﬁcantly among plant species or functional groups (Table S1,Figs. 1e3). These results suggest that biophysiological properties of plant species or functional groups, but not N amount or forms associated with chemical structure, determine their responses to atmospheric N deposition. However, in the present study we cannot deeply explain, for example, whether the aboveground responses presented are linked to belowground response (e.g. source-sink relationship for NSCs), because we did not investigate the belowground part due to restriction of destructive sampling. Further, we cannot yet explain whether responses of leaf NSCs are associated with changes in plant carbon gain (photosynthesis) or comsumption (e.g.

respiration and litter loss). We also do not know whether the responses change over the very short growing season (~3 months). Further studies will take into account of all these aspects, to better understand the grassland community responses to future atmospheric N deposition.

We conclude that the increase in aboveground biomass is a result of increased concentrations of both leaf N and NSCs under increasing N deposition, irrespective of N addition rates and N forms. Our results suggest that greater increase in mobile carbohydrate and N concentrations contributes to faster growth of grasses, relative to forbs and legumes. These results imply that future nitrogen deposition will beneﬁt grasses over other species, and thus the former may become more dominant in grasslands in northern China.

Author contributions

MHL, YJ and XGH designed the experiment; XW, GJY and MHL conducted theﬁeld sampling; MW, YMT, NNF, and JPC conducted chemical analysis; XW and FHY wrote the original manuscript and all authors reviewed and revised the manuscript.

Fig. 4.Relationships between the changes in aboveground biomass of theﬁve plant species and the changes in concentrations of nitrogen (N), soluble sugars, starch and total non-structural carbohydrates (NSCs).

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Funding

This work was supported by the Natural Science Foundation of Zhejiang Province of China (LQ20C160008), Research and Innovation Initiatives of Taizhou University (2017PY033), and the Ten Thousand Talent Program of Zhejiang Province (2018R52016).

Declaration of competing interest

The authors declare that they no conﬂicts of interest.

Acknowledgments

The authors thank the staff of Erguna Forest-Steppe Ecotone Research Station for facilitating this study, and Melissa Dawes for English language corrections.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.gecco.2021.e01507.

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