Grazing is one of the most important utilization methods of desert steppe, but its fragile ecological characteristics make soil and vegetation extremely sensitive to the disturbance of grazing livestock (Han et al., 2017; Li et al., 2018). In low productivity ecosystems, grazing results in a decrease in soil organic carbon and nitrogen pools (Holst et al., 2007; Hafner et al., 2012). Grazing makes more and more material and energy of grassland be taken away, and the absorption and utilization of plant nutrients can well reflect the growth state and internal characteristics of plants. Grassland degradation under heavy grazing will lead to the decrease of plant biomass and nutrients (Belsky 1986; Acker 1990).
The results showed that the contents of NH4 + and NO3 - in the rhizosphere soil of Stipa breviflora under heavy grazing were significantly lower than those under no grazing and light grazing, and the contents of NH4 + and NO3 - were the highest under no grazing. This is different from many studies, previous studies have shown that with the increase of grazing intensity, the nitrogen content in soil increases gradually due to the increase of feces and urine emissions of livestock (Whitehead 2000; Zaman et al., 2009). The reason for this difference is due to the different grazing forms. In this experiment, the livestock were fed in the experimental plot during the day, and were driven back to the cage at night to rest, and most of the feces and urine of livestock were left in the cage (Sun et al., 2018a). The experimental design of other studies is that livestock feed and rest from morning to night in the experimental area, and livestock excreta are distributed in the experimental area, so the soil nitrogen content is relatively high.
We found that the total nitrogen content in roots and leaves of Stipa breviflora under grazing prohibition treatment was similar to that under heavy grazing treatment, with high nitrogen content in roots and low nitrogen content in leaves. Based on the analysis of nitrogen assimilation related enzyme activities and nitrogen compound contents in the roots and leaves of Stipa breviflora under different grazing intensities, we concluded that high nitrogen (no grazing) and low nitrogen (heavy grazing) environments were not conducive to nitrogen absorption and utilization in the roots of Stipa breviflora. In this study, the nitrate nitrogen content in the root system of Stipa breviflora under grazing prohibition, moderate grazing and heavy grazing was significantly lower than that under light grazing, and the nitrate nitrogen content in the leaves of Stipa breviflora under moderate grazing and heavy grazing was also lower, which indicated that the NO3 + absorption capacity of Stipa breviflora was weakened by grazing prohibition, heavy grazing and even moderate grazing.
Nitrogen assimilation plays a crucial role in plant life activities, which directly affects the growth and development of plants (Ge et al., 2014). Nitrate reductase (NR), glutamine synthetase (GS), glutamic acid synthetase (GOGAT), glutamic oxaloacetate transaminase (got) and glutamic pyruvate transaminase (GPT) activities directly affect the nitrogen assimilation process of plants, and then affect the assimilation efficiency of nitrogen (Yu et al., 2018). The results showed that the total nitrogen content in roots and leaves of Stipa breviflora was significantly correlated with the activities of nitrate reductase, glutamate synthase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. NR is a key enzyme in the process of nitrogen metabolism, and its activity is closely related to nitrogen assimilation capacity (Zhang et al., 2008). Part of NO3 - absorbed by plant roots generates NH4 + under the catalysis of NR and NIR, and the other part is transferred to leaves. In this study, NR in the leaves of Stipa breviflora was significantly reduced under the medium grazing prohibition and heavy grazing treatment, while the NR in the root system was stronger under grazing prohibition and weak under severe grazing. This indicated that the heavy grazing treatment significantly reduced the transformation of NO3- to NH4+ in the root system of Stipa breviflora, and the conversion of NO3- to NH4+ in leaves was significantly reduced by grazing prohibition and heavy grazing, which affected the efficiency of nitrogen assimilation.
Under the catalysis of GS and GOGAT, NH4+ forms glutamic acid and glutamine, and GS and GOGAT process are carried out simultaneously. Li et al. (2018) studied the process of nitrogen assimilation in maize, and showed that the higher the activity of GS and GOGAT, the stronger the ability of nitrogen assimilation. Our research is slightly different from its results, there was a significant negative correlation between GS and total nitrogen content in roots and leaves of Stipa breviflora, that is, the higher GS activity in roots and leaves, the lower total nitrogen content in roots and leaves of Stipa breviflora. However, this is similar to the results of Fei et al. (2003), which showed that GS activity did not show the same trend with biomass and nutrient content. The reason may be related to the content of NH4+ in plant roots and leaves, because studies have shown that GS is more sensitive to low concentration of NH4+1, and GS activity will be significantly reduced under high NH4+ (Zhang & Xu, 2011). There was a significant positive correlation between GOGAT activity and total nitrogen content in roots and leaves, indicating that grazing treatment with strong GOGAT activity in roots and leaves of Stipa breviflora had higher total nitrogen content in roots and leaves, which was the same as previous studies (Wang et al., 2020; Li et al., 2018).
GOT and GPT are important transaminases in the process of nitrogen assimilation in plant roots and leaves. Wang et al. (2020) showed that with the increase of nitrogen application rate, the activities of GOT and GPT in oat leaves increased or increased first and then decreased, indicating that the suitable nitrogen addition was beneficial to the increase of GOT and GPT activities in plant leaves to a certain extent, but the GOT and GPT activities would be weakened when the nitrogen content in soil was too low or too high. This study also showed that GOT and GPT activities were significantly correlated with total nitrogen content in roots and leaves of Stipa breviflora, indicating that the higher GOT and GPT activities in roots and leaves of Stipa breviflora, the higher total nitrogen content. In this study, GOT and GPT in leaves of Stipa breviflora were relatively weak under grazing prohibition and heavy grazing, which was closely related to the low total nitrogen content in leaves of Stipa breviflora.
The process of nitrogen assimilation in plants is complex, not only involving many enzymes, but also the amount of substrate produced affects the assimilation efficiency (Zhang & Xu, 2011). Our results showed that heavy grazing significantly reduced the content of soluble protein and amino acids in roots, and significantly increased the content of amino acids in leaves, and correlation analysis showed that this was related to the changes of GS, GOT, GPT and NR activities in roots and leaves. Therefore, we believe that grazing prohibition and heavy grazing significantly affect the formation of substrate in the process of nitrogen assimilation. In our study, the proline content in roots and leaves of Stipa breviflora was higher than that of light and moderate grazing, especially in heavy grazing. Grazing can destroy the growth state and adaptability of plants, so it has been considered as the biological stress of most plant species (Shen et al., 2019). The results showed that the content of proline could reflect the stress intensity of plants to a certain extent (Ibarra Caballero et al., 1988; Rasluni et al., 2004). Therefore, we believe that grazing prohibition and heavy grazing are not conducive to the nitrogen absorption, utilization and assimilation of Stipa breviflora.