In this research, we used L. chinensis as the study species, as it is highly abundant in important grazing ecosystems. L. chinensis is a perennial species in the family Gramineae, and it is distributed in the eastern region of the Eurasian steppe (Li et al. 2015; Xu and Zhou 2006), including the outer Baikal area of Russia, the northern and eastern parts of the People’s Republic of Mongolia, the Northeast China Plain, the Northern China Plain, and the Inner Mongolia Plateau of China. The plant is highly drought tolerant and can withstand both low fertility and high pH conditions, while still producing high yields. Owing to its excellent stress tolerance, L. chinensis is found throughout a broad gradient of temperature and precipitation conditions. It is highly palatable for grazing livestock and frequently used as hay. Therefore, it is often exposed to both grazing and mowing. L. chinensis has a rhizomatous rooting system and produces clonal buds, leading to its extensive spread, through which it often forms large patches of monoculture (Bai et al. 2009). Its highly branched rhizomes lie horizontally about 10 cm under the surface of the ground. From an ecological perspective, L. chinensis is a desirable plant for use in the restoration of degraded grassland because its rapid propagation and rhizomatous net can reduce soil erosion and desertification in arid areas, such as those of northern China (Liu and Han 2008).
Field study: plant and soil effects of grazing
The field site of our study is located at the Xilingol Grassland Ecosystem Research Station (XGERS) in the Xilin River catchment (43°38′ N, 116°42′ E) of the Inner Mongolia Autonomous Region, P.R. China, which has a semiarid continental climate (Yao et al. 2010). According to long-term monitoring (1982–2016), the climate is characterized by an annual precipitation of approximately 320 mm, with more than 70% falling from May to August, which coincides with the highest temperatures (Li et al. 2015). The mean annual temperature is 1.01 °C, ranging from − 18.8 °C in January to 21.6 °C in July. The major soil types of this region are calcic chestnuts and calcic chernozems (Gong et al. 2008). The vegetation is a temperate steppe, and the dominant species are L. chinensis and Stipa grandis (Li et al. 2015).
In this study, the field experiment consisted of two treatments: grazing exclusion and heavy grazing by livestock. The grazing plot, which was about 200 ha in area, has experienced year-round grazing by sheep and goats for more than 40 years. The stocking rate of approximately 3.0 sheep units ha− 1 is substantially higher than the recommended stocking rate of 1.5 sheep units ha− 1 (Li et al. 2015; Ren et al. 2017). Since 1983, the grazing exclusion plot adjacent to the grazing area has been fenced off for the purpose of long-term ecological observation and research by XGERS. Using the paired sampling method, five 10 m × 20 m replicated subplots were established in treatments of both grazing exclusion and heavy grazing along the pasture fence. The paired subplots were randomly allocated within 30 m of each other along the pasture fence.
The plant and soil sampling was conducted in the ten selected subplots of grazing exclusion and heavy grazing on August 15, 2016, corresponding to the peak growing season of L. chinensis. In each subplot, three 1 m × 1 m quadrats were randomly established for sampling. Each quadrat was divided into three equal parts. In the first part of the quadrats, the green leaves of L. chinensis individuals were collected in order to estimate leaf N resorption efficiency (Lü et al. 2012). To analyze the potential influence of grazing on leaf N resorption, we estimated N concentrations from relatively young (upper) and relatively old (lower) leaves in the second part of each quadrat. At the same time, we sampled soil associated with L. chinensis following the same plant sampling scheme using a soil auger (diameter = 7 cm, depth = 20 cm). In the third part of the quadrats, we collected about 15 senesced leaves of L. chinensis on November 15, 2016, corresponding to the non-growing season.
After the field sampling, plant traits, such as plant height, dry matter content (DMC), and aboveground biomass, were measured at the individual scale in the laboratory. The fresh weights of L. chinensis leaves were measured and were then oven-dried at 65 °C for 48 h to determine the DMC. Using a mechanical micromill, the green and senesced L. chinensis leaves were then ground. The ground samples were passed through a 0.5-mm mesh sieve. Soil samples were air-dried at room temperature for 20 days. The soil samples were then ground, homogenized, and passed through a 2-mm mesh sieve after removing fine roots and stones. Using the Kjeldahl method, total concentrations of plant and soil N were measured (Wang et al. 2018). The soil organic matter content was analyzed using dichromate oxidation (Li et al. 2019b). In addition, available soil N soil was photometrically measured by a continuous flow analyser (SAN Plus, Skalar, Netherlands) from on-site KCl extractions using the fresh soil (Geng et al. 2017).
Growth chamber study 1: test for grazing-related plant legacy effects
The growth chamber study was a continuation of our field grazing experiment. The method of asexual reproduction by rhizome buds of L. chinensis was used to determine how long-term heavy grazing influences plant legacy effects on N processing. In each quadrat of our field site, we collected more than five dormant buds of L. chinensis in March 2017 during peak production of rhizome buds (from November 2016 to March 2017). In total, more than 15 buds (more than five buds from each of three replicated quadrats) were prepared corresponding to each of the ten field subplots (five grazing subplots and five ungrazing subplots). At this stage, all the buds were at the initial development state. In the laboratory, the associated rhizomes with buds were cut into 2-cm lengths to ensure the offspring had similarly proportioned sources of nutrients from maternal rhizomes. The clonal offspring were cultivated in a hydroponic container with Hoagland nutrient solution (15 cm × 15 cm × 12 cm) (Ren et al. 2017). In offspring groups consisting of samples from both grazing exclusion and heavy grazing, we had four replicates (four randomly selected from the five field sampling subplots) for each experimental container. In each of the hydroponic containers, 12 individuals of L. chinensis clonal offspring were retained after excluding other dead buds or plants that grew inconsistently. Therefore, 96 experimental plant individuals were grown (2 maternal treatments × 4 containers × 12 buds). In our plant growth chamber (Percival Scientific, Inc., Perry, IA, USA), the lights remained on for 14 h day− 1 with a 25 °C daytime temperature, 15 °C nighttime temperature, and relative humidity of 70–80%. We changed the Hoagland nutrient solution every five days. During the experiment, we measured plant height every seven days, which was used as the indicator of clonal plasticity induced by plant legacy effects. After 65 days of growth, half of the plants were sampled for the purpose of measuring phenotypic traits, aboveground biomass, and N concentrations after they were oven-dried at 65 °C for 48 h.
Growth chamber study 2: plant legacy effects on N metabolism
Using the plant materials of growth chamber study 1, plant samples for the analysis of N-related enzyme activity and gene expression were sampled after 65 days of cultivation. The mature plants were immediately frozen in liquid N2 and then stored in an Ultra-low Temperature Freezer (Thermo Fisher, Waltham, MA, USA) at -80 °C. The activities of nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase of leaves and roots were determined with a Nitrate Reductase Kit (NR-1-Y, Comin), Nitrite Reductase Kit (NIR-1-G, Comin), Glutamine Synthetase Kit (GS-1-Y, Comin), and Glutamate Synthase Kit (GOGAT-1-Y, Comin) following the manufacturer’s instructions. In addition, the expression levels of three N-related genes (NRT1.1, NRT1.2, and NRT1.7) in leaves, which had been preliminarily confirmed by our transcriptome data, were measured using real-time quantitative PCR (qPCR). Total RNA levels were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Full-length cDNA was then reverse transcribed using a cDNA synthesis kit (RR047A, TaKaRa, Dalian, China). For each gene, qPCR was performed according to the manufacturer’s instructions (4472908, Applied Biosystems, Foster City, CA, USA), and all the genes were assayed with three independent biological replicates. The Actin gene (GenBank accession number, HM623326.1) was used as an internal reference. The reaction conditions were 30 s at 95 °C, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primer sequences are provided in Supplementary Table S1.
Growth chamber study 3: plant legacy effects on root foraging
In order to investigate the potential mechanism of root foraging behavior underlying N uptake, we studied the foraging behavior of grazed and ungrazed plant offspring roots in a mesocosm study. Plants from two maternal source treatments (OG+, clonal offspring of plants that had experienced long-term herbivore grazing; OG-, clonal offspring of plants that had not experienced grazing) exposed to a soil N patch were distributed in a randomized, complete block design with eight replicates. The background soil was collected from natural grassland near XGERS and passed through a 5-mm mesh sieve to remove the fine roots and stones. The pots (15-cm diameter and 18-cm deep) were split into two parts by polyethylene barriers. In order to create N heterogeneity, half of each pot was filled with N rich soil by adding 150 mg NH4NO3, whereas the other half was filled with original background soil. After 10 days of pre-cultivation in hydroponic nutrient solution, the asexual offspring of L. chinensis were transplanted into pots using the root-splitting method (Sun et al. 2016). After 45 days of asexual offspring establishment, the root length and root biomass were measured both in the N-rich and N-poor parts to assess the sensitivity of clonal offspring of grazed and ungrazed L. chinensis to soil N patches.
Calculation and statistical analysis
Significant differences in plant phenotypic traits, physiological traits, and gene expression levels between the experimental groups were assessed by one-way ANOVA. Before the analyses, the phenotypic traits, including plant height, DMC, and individual biomass, were averaged from all the L. chinensis plants of each quadrat in the field grazing experiment and of one hydroponic container in the growth chamber study. In addition, the relative levels of the three N-related genes from the qPCR analysis were log-transformed before analysis.
Leaf N resorption efficiency (NRE) was defined as the proportion of N in mature leaves that was resorbed during senescence (Lü et al. 2015). In the field experiment, NRE was calculated as
NRE = (1 - Nsenesced / Ngreen) × 100%, (1)
where Nsenesced and Ngreen are the N concentrations of senesced L. chinensis leaves in November and green L. chinensis leaves in August, respectively. The N concentrations of L. chinensis leaves were expressed on a dry mass basis.
In this study, plant N accumulation (Naccumulation), N uptake efficiency (NTE), N utilization efficiency (NUE), and N utilization index (NUI) were calculated to assess the N use strategies. These calculations were as follows:
Naccumulation = BM × Nconcentration, (2)
NTE = Naccumulation / Nsupply × 100%, (3)
NUE = BM / Naccumulation, (4)
NUI = BM × NUE. (5)
Above, BM is the total biomass of one hydroponic container, and Nsupply is the total quantity of N supply in Hoagland nutrient solution.
The plasticity index (PI), defined as the responses of plant height to grazing induced plant legacy effects (Li et al. 2015), were calculated as
PI = (HOG− – HOG+) / HOG− × 100%, (6)
where HOG− is the plant height of the clonal offspring of plants that had not experienced grazing and HOG+ is the clonal offspring of plants that had experienced long-term livestock grazing.
The response ratios (RRs) of L. chinensis root traits, including root length and biomass, to soil N heterogeneity in the root-splitting experiment were calculated as
RR = ln(TN+ / TN−), (7)
where TN+ and TN− are the root traits of plants from the N-rich and N-poor patches, respectively.