Effects of long-term warming on microbial nutrient limitation of soil aggregates on the Qinghai-Tibet Plateau

In the present study, we used open-top chamber experiments to simulate warming in an alpine meadow and an alpine shrubland on the Qinghai-Tibet Plateau, and we measured the C, N, and P-acquiring enzyme (β-1, 4-glucosidase, BG; leucine aminopeptidase, LAP; β-N-acetylglucosaminidase, NAG; alkali phosphatase, AP) activities and their stoichiometry to understand how warming affects microorganism-limiting mechanisms in soil aggregates. our study increased our understanding of the effects of warming on microbial nutrient utilization and restriction patterns in soil aggregates. the microorganisms on the a long-term The showed that long-term warming treatment signicantly decreased organic carbon (SOC) and total nitrogen (TN) concentration of large macroaggregates (LMGA) and small macroaggregates in alpine meadow, but signicantly increased SOC concentration of LMGA


Introduction
Loading [MathJax]/jax/output/CommonHTML/jax.js Starting with the Industrial Revolution, greenhouse gas emissions have been causing a continuous increase in global temperatures, and a temperature rise of 2-4.5°C has been predicted by the year 2100 (O'Neill et al., 2017; Rogelj et al., 2012). The Qinghai Tibet Plateau possesses the world's highest and largest alpine grasslands, and its fragile ecological environment is highly vulnerable to climate change (Lehnert et al., 2016). Global warming impacts not only the soil environment, including soil temperature and moisture (Brzostek et al., 2012;Nie et al., 2014), but also the length of the growing season (Post et al., 2009) and species composition of plant communities (Saxe et al., 2001). In order to better understand the response of the Tibetan Plateau to future climate change, more mechanism characteristics of ecological structure and function need to be considered.
Soil microorganisms, the driving forces of the biogeochemical cycle and energy ow in ecosystems (Sinsabaugh and Biochemistry, 2010), are very sensitive to changes in the living environment. However, microorganism growth and development are usually limited by nutrient resources (Ekblad and Nordgren, 2002). Changes in external conditions (such as land-use changes, nitrogen deposition, and global warming) often affect the nutrient limitation patterns of microorganisms by changing the state and concentration of soil nutrients (Chen et al., 2018;Chen et al., 2019;Wang et al., 2014). For example, Chen et al., (2018) found that N addition aggravated microbial C limitation, and Chen et al., (2019) found that cropland conversion alleviated microbial C limitation, which was due to the elevated soil carbon concentration. Previous studies have shown that warming changed the state and stoichiometry of soil C, N, and P by regulating the growth of plants and microorganisms in the Tibetan Plateau Zhang et al., 2015), which may directly change the nutrient limitation pattern of microorganism.
Soil enzymes play a key role in decomposing organic matter and determining the availability of soil nutrients (Meng et al., 2020). Microorganisms decompose soil organic matter by releasing the C-, N-, and P-acquiring enzymes to meet their corresponding resource demands (Qiao et al., 2019). Previous studies have shown that the soil enzyme activity responses to warming are diverse (Bell et 2020) found that short-term warming decreased carbon-degrading enzymes and increased C:P enzyme ratios, as well as proposed that warming decreased microbial C limitation but increased microbial P limitation in the alpine timberline of the eastern Tibetan Plateau. Therefore, we believe that the study of soil enzyme activity and its stoichiometry can better reveal the effect of warming on microbial relative C, N, and P limitations in the Qinghai Tibet Plateau.
However, to our knowledge, there is a lack of research on the effects of experimental warming on the relative C, N, and P limitations of microbial processes at the soil aggregate level in Qinghai Tibet Plateau. Soil aggregates are important factors that affect soil physical and chemical properties (such as soil porosity, soil bulk density, water-holding capacity, and soil erosion resistance), thereby shaping the basic soil physical structure (Deng et al., 2018;Zhu et al., 2017). A stable soil aggregate structure provides microorganisms with a suitable living environment by adjusting the ow of water and oxygen (Six et al., 2004 physical protection of soil organic matter and affect the availability of substrates to microorganisms, which will affect the potential microbial nutrient limiting mechanism. Liu et al. (2021) suggested that long-term warming reduced the SOC and TN concentrations, substrate availability to microbes, and enzyme activity, especially in macroaggregates. Thus, microbial thermal responses in macroaggregates are more sensitive, which may also be the main way of soil nutrient loss under warming . The stronger nutrient limitation in microaggregates may also be compensated by the longer turnover time of microbial biomass under long-term warming (Bailey et al., 2012). In summary, soil aggregate size mediates microbial climate change feedbacks, and exploring the impact of climate warming on the nutrient limitation mechanism of soil aggregates in the Qinghai-Tibet Plateau is of great signi cance for further understanding the response of alpine grassland ecosystems to warming.
In the present study, we investigated the distribution characteristics of soil aggregate nutrients and enzyme activities in an alpine meadow and an alpine shrubland on the Qinghai-Tibet Plateau under a warming treatment, using chemometric knowledge to explore the resource limits of microorganisms at the aggregate level. We hypothesized that (1) long-term warming treatment would reduce soil nutrient concentration and soil enzyme activity, and (2) microbial nutrients limitation would increase and be the strongest in MIGA as a consequence of warming.

Experimental site
The study site was located at the Haibei Alpine Meadow Ecosystem Research Station (101° 12' E, 37° 30' N, 3200 m a.s.l.), in the northeastern part of the Qinghai-Tibet Plateau (Fig. 1). The area has a typical continental monsoon-type climate. The summers are short and cool, and the winters are long and cold (Ji et al., 2017;Jiang et al., 2016). The average annual temperature is -1.7 ℃, annual extreme maximum temperature is 27.6 ℃, annual extreme minimum temperature is -37.1 ℃, and annual precipitation range is 426-860 mm, 80% of which is distributed in the growing season from May to September. The average annual sunshine duration is 2462.7 hours, with 60.1% total available sunshine (Zhao and Zhou, 1999). The main soil type is Mollic-Cryic Cambisol (Zhao and Zhou, 1999

Soil sampling and analysis
Samples of undisturbed soil were taken from warmed plots and control plots on August 10, 2018 by collecting the surface layer (0-5 cm) of bulk soils in quadruplicate from each plot using a soil core sampler. Since OTCs showed the warming effect at a depth of 0-12 cm and the warming effect decreases with the depth of the soil layer , the surface soil of 0-5 cm was selected in the sampling. Subsequently, the samples from each plot were mixed to form a composite sample. Litter and debris were removed before sampling. We carefully transported the samples back to the laboratory to avoid damaging the soil physical structure. In all soil samples, we used the 'optimal moisture' method to separate the aggregates (Bach and Hofmockel, 2014;Dorodnikov et al., 2009;Mendes et al., 1999). First, soil samples were placed in a 4°C constant-temperature environment to dry until reaching approximately 10% gravimetric water content. The samples were then gently sorted by hand to below 8 mm in size and shaken three times for 2 min using a mechanical shaker to partition the aggregate sizes. Soil samples were then sieved into three grades using stacking sieves (2 and 0.25 mm): > 2 mm (large macroaggregates, LMGA), 2-0.25 mm (small macroaggregates, SMGA), and < 0.25 mm (microaggregates, MIGA) (Nie et al., 2014). A part of each composite soil sample was placed in a refrigerator at 4°C for two weeks to determine soil enzyme activity, and the rest was air-dried to measure other physicochemical properties.

Soil physicochemical properties and enzyme analysis
Soil organic C (SOC) was assayed by dichromate oxidation (Kalembasa et al., 2010). Total N (TN) was assayed using the Kjeldahl method (Bremner and Mulvaney, 1982). Total P (TP) was measured colorimetrically using the ammonium molybdate method (Schade and Levine, 2003).
We determined the activity of β-1,4-glucosidase (BG), leucine aminopeptidase (LAP), β-Nacetylglucosaminidase (NAG), and alkaline phosphatase (AP) using a modi ed version of standard uorometric techniques (Chen et al., 2020; Wu et al., 2020) ( Table 1). In brief, 3 g soil samples were placed in 125 mL of Tris buffer (50 mM, pH = 8.0) and homogenized. The samples were placed in a 96-well microplate with eight repeating groups in each column. After the microplate was loaded, it was shaken, mixed well, and placed in a 25°C incubator for 0.5, 2, or 4 h. We then used a microplate reader at 365 nm for excitation and 450 nm for emission to measure the amount of uorescence, and enzyme activity was expressed in nmol·g − 1 ·h − 1 . SOC, TN, TP, and enzyme activities were analyzed by one-way analysis of variance (ANOVA) to assess the effect of long-term warming treatment on their distribution in soil aggregates. Duncan's tests at p < 0.05 were performed for multiple comparisons. All statistical analyses were performed using SPSS 20.0 (IBM SPSS, Chicago, IL, USA), and Origin 9.0 (Origin Lab Corporation, Northampton, MA, USA) was used to prepare graphs.
Vector analysis of enzymatic stoichiometry was used to measure resource limitation (Chen et al., 2018; Moorhead et al., 2013). This method is based on the relative levels of C, N, and P-acquiring enzyme activity to explore the limitations of microbial resources by using the following equations: Relatively longer vector L indicated greater C limitation; the vector A < 45° and > 45° indicated N and P limitation, respectively. When the vector A < 45°, smaller angle indicates stronger N limitation of microbial resources. Similarly, when the vector A > 45°, greater angle indicates stronger P limitation of microbial resources.

SOC, TN, TP, and enzyme activities
In the alpine meadow, the SOC concentration under the warming treatment was lower than that under the control treatment, and there was a signi cant difference in LMGA and SMGA between the two treatments ( Fig. 2). However, in the alpine shrubland, the SOC concentration under the warming treatment was higher than that under the control treatment, and there was a signi cant difference in LMGA between the two treatments. In the alpine meadow, the TN concentration under the warming treatment was lower than that under the control treatment, and there was a signi cant difference in LMGA and SMGA between the two treatments. In the alpine shrubland, there was no signi cant difference in TN concentration between the warming treatment and the control treatment. In the alpine meadow, the concentration of SOC and TN decreased with the increase in soil aggregate size, and the concentration of MIGA was signi cantly higher than that of LMGA. In the alpine shrubland, SOC and TN concentrations did not change with the changes in soil aggregate size. TP concentration in both the alpine meadow and alpine shrubland was less affected by the long-term warming treatment.
In most cases, the warming treatment had no signi cant effect on enzyme activity, except for AP and LAP of MIGA and BG of SMGA in the alpine shrubland (Fig. 3). In the alpine meadow, BG activity was signi cantly higher in MIGA than in LMGA and SMGA. In the shrubland, BG, NAG, and AP activities in MIGA were signi cantly higher than those in LMGA and SMGA. BG, LAP, NAG, and AP activities increased as aggregate size decreased and were higher in the alpine meadow than those in the shrubland. In the alpine meadow, compared to the control treatment, the long-term warming treatment signi cantly reduced the C:P ratios of LMGA and SMGA and the N:P ratios of SMGA and MIGA, whereas it had no signi cant effect on the C:N ratios (Table 2). In this habitat, the C:P and N:P ratios increased as the soil aggregate size decreased, and MIGA size was signi cantly larger than LMGA size. In the shrubland, compared to the control treatment, the warming treatment signi cantly increased the soil aggregate C:N ratios and the C:P ratios of LMGA and SMGA. The long-term warming treatment had no signi cant impact on enzyme stoichiometry in the alpine meadow ( Table 3). The enzymatic C:P and N:P activity ratios were decreased in the alpine shrubland under the warming treatment compared to those under the control treatment, and there were signi cant differences in LMGA between the warming and control treatment. Soil aggregate size had no signi cant effect on enzyme stoichiometry in both habitats.

Discussion
Effects of warming treatment on nutrients and enzyme activities in soil aggregates shown that the nutrient turnover rate in large aggregates is higher than that in small aggregates (Jastrow, 1996;Six et al., 2000). In alpine meadow, the results showed that the concentrations of SOC and TN in LMGA and SMGA were signi cantly decreased under warming treatment, but warming had no signi cant effect on them in MIGA. This indicated that the carbon and nitrogen mineralization rates of large aggregates was more sensitive to warming than those of microaggregates. However, in the shrubland, we found that SOC in the warming treatment group was higher than that in the control group, and there was a signi cant difference in LMGA between the two groups. During sampling, we found warming signi cantly decreased the evenness index, diversity index and abundance index in alpine shrubland, but increased above-ground biomass (Table   s1). It is possible that the warming treatment reduced the soil moisture (Nie et al., 2014), and the strong Loading [MathJax]/jax/output/CommonHTML/jax.js competition with shrubs resulted in the lower abundance of herbaceous plants. The increased aboveground biomass is mainly shrub biomass, and the refractory shrub litter promoted the accumulation of surface soil nutrients. In the present study, as TP was mainly affected by the soil parent material; warming treatment had no signi cant effect. In the alpine meadow, SOC and TN concentrations decreased as soil aggregate size increased. Compared to LMGA, MIGA may provide better physical protection and has more stable SOC and TN concentrations (Six et al., 2001).
Soil extracellular enzyme activity can re ect the response of microorganisms to their nutrient requirements . The greater speci c surface area of smaller aggregates than that of larger ones facilitates the attachment of microorganisms on their surface (Amato and Ladd, 1992;vanGestel et al., 1996). In addition, smaller pore sizes in MIGA than those in LMGA provide better physical protection for microorganisms, thereby enabling them to stay in the soil for a longer time (Zhang et al., 2013).

Effects of warming treatment on microbial resource limitation
Some studies have indicated that warming can increase dissolved organic carbon concentration in the soil solution, which in turn decreases the microbial C limitation (Luo et al., 2009). However, our results demonstrated that the warming treatment had no signi cant effect on microbial C limitation in the alpine meadow and shrubland. This may be because of the balance between microorganism carbon demand and the input of soil organic carbon during the long-term warming treatment. At the same time, enzyme analysis results proved this point, which showed that warming had no signi cant effect on BG (C-acquiring enzyme) activity, except in SMGA in the shrubland. The vector L decreased with increase in soil aggregate size, and MIGA was signi cantly higher than LMGA in the alpine shrubland. Soil aggregate structure affects microbial activities as uxes of water and oxygen (Six et al., 2004), which can affect the accumulation and distribution of soil nutrients (Jastrow et al., 2007). It can also provide physical protection to prevent the rapid decomposition of soil organic carbon, and the level of protection depends on aggregate size (Pulleman and Marinissen, 2004). Jastrow et al. (2007) reported that the protection of SOC by MIGA is greater than the protection of SOC by LMGA, and soil organic matter (SOM) is more recalcitrant in MIGA than in LMGA.
Researchers have also found that the water-soluble carbon and active carbon in macroaggregates were signi cantly higher than those in micro-aggregates (Jha et al., 2012).
Our results showed that the vector A was greater than 45°, suggesting that microbial P limitation was widespread in the investigated alpine meadow and shrubland. We also found that lnBG:lnAP and ln(NAG + LAP):lnAP were slightly below 1.0, which also demonstrated that the P acquisition enzyme activity was relatively higher than the C and N acquisition enzyme activities. The analysis of the second national soil survey showed that the available phosphorus content in the Qinghai-Tibet Plateau is generally lower than the national average (Wang et al., 2008). This may be related to the cold climate of the Qinghai-Tibet Plateau and low rates of P-containing primary mineral weathering (Rui et al., 2012). Compared to the control treatment, the long-term warming treatment signi cantly exacerbated the microbial P limitation in the alpine shrubland, which was indicated by higher vector A. It shows that soil microbial P limitation in shrubland is more sensitive to warming treatment. It is possible that warming promoted the absorption of soil phosphorus by plants, which decreased the concentration of soil available phosphorus, and strengthened the competition between plants and microorganisms for phosphorus resources (Gong et al., 2020). Warming can increase the accumulation of P in plant biomass (Rinnan et al., 2008) and strengthen the P competition between soil microorganisms and vegetation. In addition, we found that the long-term warming treatment intensi ed resource competition among plants and led to a decrease in herb biomass. Branches and leaves of shrubs are more di cult to decompose than herbs (Brigham et al., 2018), resulting in weakened nutrient cycling and low available P content.

Conclusions
In the present study, we investigated the distribution characteristics of nutrients, enzyme activities, and ecological stoichiometry of soil aggregates in an alpine meadow and alpine shrubland, and explored the resource limits of soil aggregate microorganisms on the Qinghai-Tibet Plateau under a long-term warming treatment. The results showed that long-term warming treatment signi cantly decreased soil organic carbon (SOC) and total nitrogen (TN) concentration of large macroaggregates (LMGA) and small macroaggregates (SMGA) in alpine meadow, but signi cantly increased SOC concentration of LMGA in alpine shrubland. The SOC and TN concentrations of alpine meadow increased with the decrease of soil aggregate size and the concentrations in microaggregate (MIGA) were signi cantly higher than those LMGA. Enzyme activity increased as aggregate size decreased and was not signi cantly affected by the warming treatment. Enzyme stoichiometry demonstrated that alpine meadows and shrublands on the Qinghai-Tibet Plateau are mainly microbial P limitation relative nitrogen, and the long-term warming treatment exacerbated P limitation, which had signi cant differences in shrubland. At the same time. The long-term warming treatment had no signi cant effect on the C limitation in the alpine shrubland and alpine meadow, but the soil aggregate size