Effects of Soil Nutrients on Plant Nutrient Traits in Natural Pinus tabuliformis Forests

In light of global warming, the interaction between plant nutrient traits and soil nutrients is still unclear. Plant nutrient traits (e.g., N and P) and their stoichiometric relationships (N/P ratio) are essential for plant growth and reproduction. However, the specific role of soil nutrients in driving variation in plant nutrient traits remains poorly understood. Fifty natural Pinus tabuliformis forests were used as the research object to clarify the interaction between plant nutrient traits and soil nutrients. We show that: (1) The Nmass, Pmass and N/P ratios of leaves were significantly higher than those of roots. The N/P ratio of both leaves and roots was less than 14. (2) Leaf nutrient traits showed diverse relationship patterns with root nutrient traits throughout the growing period. Significant changes were found in root nutrient PC2 (the second principal component of root nutrient traits) and leaf nutrient PC1 (the first principal component of leaf traits), and non-significant changes were found in other relationships between leaf and root traits (p > 0.05). Root nutrient traits explained 36.4% of the variance in leaf nutrient traits. (3) With the increase in soil nutrient PC2 (related to N), leaf PC2 (related to N) showed a significant trend of first decreasing and then increasing (p < 0.05). Only the soil Nmass was significantly correlated with the leaf Nmass (p < 0.05), which demonstrated that the growth and survival of Pinus tabuliformis forests were mainly affected by N-limitation.


Introduction
Nutrient traits refer to traits related to nutrient characteristics (e.g., N mass and P mass ), reflecting the survival strategies of plants in response to global warming, and are widely used in ecology [1][2][3]. Nitrogen (N) and phosphorus (P) are basic components of plant genetic material and nutrients, and their stoichiometric relationships significantly influence the process of plant growth and reproduction [4,5]. As a major limiting element, N is a fundamental component of enzymes [4]. Meanwhile, phosphorus drives the generation and maintenance of proteins and is also limiting in most environments [6]. The absence of nitrogen and phosphorus in leaves will affect the formation of chlorophyll, further reduce the productivity of forest communities and regulate carbon cycling [3]. The ratio of nitrogen to phosphorus (N/P) in plants reflects environmental factors, especially the nutrient supply of soil-to-plant growth [7,8]. It can clarify which elements restrict the plant's productivity. In other words, in a habitat where P is scarce and N is relatively abundant, plant N/P is relatively high, while in the habitat where N is scarce and P is relatively rich, plant N/P is relatively low and plant P content is significantly increased [3]. Soil nutrient limitation not only affects the nutrient structure of species but also affects the composition of community species and the direction of community succession [9].
Some studies have found that climatic factors may have a certain impact on plant nutrient traits [10]. However, as the direct living environment of plants, soil provides the necessary water and nutrients for plants to survive [11]. Therefore, soil nutrient factors may play a more critical role in shaping plant nutrient trait differences. However, the specific interaction between soil nutrients and plant nutrients remains unclear.
Complex feedback regulation mechanisms exist between plant nutrients and soil nutrients [12]. For example, litter in nutrient-rich soils will introduce higher nutrients, which release large amounts of nutrients after decay, thus maintaining a higher soil fertility level. In nutrient-poor soils, plants naturally produce less litter, and their decay progresses very slowly, which further leads to soil barrenness [4,11]. N and P are important components of soil nutrients, and it is unclear whether their performance is consistent with that of plant nutrient feedback and which nutrient trait plays a more important role in plant-soil nutrient feedback.
Plants with different life forms occupy different plant-soil nutrient feedback [13]. The nutrient profiles of leaves vary significantly between different life forms due to differences in their survival strategies [2]. This plant response feedback on soil nutrient supply reflects a nutrient trade-off in plant growth and development and reflects the survival strategies adopted by plants in coping with survival pressure [14]. Based on global data, Wright et al. [15] found that the leaf nitrogen and phosphorus contents of shrubs were significantly higher than those of trees. Plants with long-living leaves have low N and P contents [2] and, thus, tree species can adapt to a living environment with low nutritional status.
In recent years, an increasing number of studies have attempted to explore the correlations between traits of different organs from the perspective of plant functional traits. Relevant studies not only help to understand the mechanisms of interaction between plant traits [4] and the utilization and allocation of resources during plant growth [16] but also have important significance in further predicting the response of plants to environmental changes. Previous studies on plant nutrient traits (e.g., N, P) often focused on aboveground organs, and few studies were conducted on roots [17]. Exploring the difference in nutrient traits between aboveground and underground organs can help us to better understand the nutrient allocation strategies of plants as well as the plant-soil nutrient feedback [18].
Songshan Nature Reserve preserves the only natural Pinus tabuliformis forest in North China. Pinus tabuliformis plays an extremely important role in resisting wind and sand, conserving water sources and purifying air, etc. The shrub species are Syinga reticulata var. Mandshurica, Corylus mandshurica and Euonymus verrucosus. Pinus tabuliformis is the constructive species in Songshan Nature Reserve. We aim to explore the relative effects of soil nutrients on plant nutrient traits based on the plant nutrient trait data (e.g., N mass , P mass ) of roots and leaves and soil nutrient data collected from 50 natural Pinus tabuliformis forests. We proposed the following hypotheses: (1) There exists a significant difference between roots and leaves in nutrient traits (e.g., N mass , P mass ). (2) Soil nutrient factors are better at explaining the variation in root nutrient traits than leaf nutrient traits.

Results
The N mass ( Figure 1A), P mass ( Figure 1B) and N/P ratios ( Figure 1C) of leaves were significantly higher than those of roots (Table 1). We also found that the phosphorus content of trees was significantly higher than that of shrubs. However, there was no significant difference (p > 0.05) in nitrogen content between trees and shrubs ( Figure S1).
The first two principal components (PC1 = 56.97%; PC2 = 41.54%) can explain 98.51% of the soil nutrient variation. The N/P ratio and PC1 showed a positive correlation. However, the N mass , P mass and PC1 showed a negative correlation (Figure 2A). The first principal component mainly represents the components related to nutrient restriction. The second principal component mainly represents the components related to nitrogen.
The first two principal components (PC1 = 53.61%; PC2 = 44.22%) can explain 97.83% of the plant nutrient variation. Both the nutrient function traits of roots and the nutrient traits of leaves were positively correlated with the second principal component ( Figure 2B). The first principal component mainly represents the components related to nutrient restriction. The second principal component mainly represents the components related to nitrogen.  The first two principal components (PC1 = 56.97%; PC2 = 41.54%) can explain 98.51% of the soil nutrient variation. The N/P ratio and PC1 showed a positive correlation. However, the Nmass, Pmass and PC1 showed a negative correlation ( Figure 2A). The first principal component mainly represents the components related to nutrient restriction. The second principal component mainly represents the components related to nitrogen.     The first two principal components (PC1 = 56.97%; PC2 = 41.54%) can explain 98.51% of the soil nutrient variation. The N/P ratio and PC1 showed a positive correlation. However, the Nmass, Pmass and PC1 showed a negative correlation (Figure 2A). The first principal component mainly represents the components related to nutrient restriction. The second principal component mainly represents the components related to nitrogen.  From the results of the generalized additive models (GAMs), it is clear that the investigated leaf traits showed diverse relationship patterns with root nutrient traits throughout the growing period (Table 2; Figure 3). Significant dynamic changes were found in the root nutrient PC2 and leaf PC1, and non-significant dynamic changes were found in other relationships between the leaf and root traits. Root nutrient traits explained 36.4% of the variance in leaf nutrient traits (Table 2; Figure 4).
The soil nutrient factors had non-significant effects on the root nutrient traits (Table 2; Figure 5). The soil nutrient factors had significant nonlinear effects on the leaf nutrient traits (p < 0.05). With the increase in soil nutrient PC2 (related to N), leaf PC2 (related to N) showed a significant trend of first decreasing and then increasing (p < 0.05). Generally speaking, soil nutrients explained 25% of the variance in the leaf nutrient traits. 2B). The first principal component mainly represents the components related to nutrient restriction. The second principal component mainly represents the components related to nitrogen.
From the results of the generalized additive models (GAMs), it is clear that the investigated leaf traits showed diverse relationship patterns with root nutrient traits throughout the growing period (Table 2; Figure 3). Significant dynamic changes were found in the root nutrient PC2 and leaf PC1, and non-significant dynamic changes were found in other relationships between the leaf and root traits. Root nutrient traits explained 36.4% of the variance in leaf nutrient traits (Table 2; Figure 4).   The soil nutrient factors had non-significant effects on the root nutrient traits (Table 2; Figure 5). The soil nutrient factors had significant nonlinear effects on the leaf nutrient traits (p < 0.05). With the increase in soil nutrient PC2 (related to N), leaf PC2 (related to N) showed a significant trend of first decreasing and then increasing (p < 0.05). Generally speaking, soil nutrients explained 25% of the variance in the leaf nutrient traits.    N) showed a significant trend of first decreasing and then increasing (p < 0.05). Generally speaking, soil nutrients explained 25% of the variance in the leaf nutrient traits.

Discussion
We found that the N mass and P mass of leaves were significantly higher than those of roots. Leaves are the main organ for photosynthesis, and N mass and P mass are closely related to protein synthesis [2]. Plant roots cannot participate in photosynthesis due to lack of chlorophyll. Therefore the N mass and P mass of plant roots are significantly lower than those of leaves [19][20][21][22][23][24][25].
We also found that the P mass of tree leaves was significantly higher than that of shrub leaves. Plants of different life forms have unique niches and different resource utilization strategies for light, temperature and water under environmental pressure [2,[25][26][27][28][29][30][31][32]. However, there was no significant difference in N mass between tree and shrub leaves. The "community construction theory" based on nutrient traits explains that, in a local community, competition may lead to divergence between traits, but habitat screening may lead to the convergence of traits [33]. Often, the habitat selection effect causes different species to form more consistent characteristics so as to adapt to the same environment [3].
There exists a tradeoff between the nutrient traits of different plant organs [15]. N mass and P mass , in plant leaves, are mainly used for photosynthesis, while those of roots are mainly used for underground ecological processes so as to adapt to adverse environments. Therefore, when plants absorb nutrients from the soil, they will balance the nutrients according to their environment [34]. When plants are in an environment with sufficient resources (sufficient light, water, and heat), they will use more nutrients for photosynthesis to maximize resource utilization and facilitate plant growth and reproduction [26]. When plants are affected by osmotic stress, they will use more nutrients in underground processes (e.g., rooting) to avoid the threat [9].
In the past century, aboveground ecology has attracted extensive attention. However, the ecological links between aboveground and underground components remain unclear [2,3]. This knowledge gap hampers our ability to understand and predict the comprehensive responses of an ecosystem to environmental stresses [2,3]. Increasing evidence emphasizes that the importance of strong interactions between aboveground and underground components in regulating ecosystem multifunctionality and responses to global change [19][20][21][22][23][24][25][26].
Plants and soil participate in the global material cycle together, existing in a close relationship [27]. Plants absorb nitrogen and phosphorus from the soil through their roots and return them to the soil in the form of litter [20]. Therefore, there is a feedback relationship between soil and plant nutrients [3]. The aboveground element characteristics of plants are usually related to the soil nutrient content. As the main substrate for plant growth, soil contains organic matter, nitrate nitrogen and ammonium nitrogen, which are decomposed to continuously provide essential nutrients for the normal physiological activities of plants. This enables the soil and plant to achieve and maintain a balanced element ratio through the dynamic exchange of nutrient supply and demand [28][29][30][31][32]. We found that only soil N mass was significantly correlated with leaf N mass , offering evidence that the growth and development of Pinus tabuliformis forests were mainly limited by the supply of soil N mass , and P mass was not the key element limiting factor.

Nutrient Trait Data
More than 10 mature and well-developed Pinus tabuliformis trees were selected from each plot to collect fresh (one-ye ar-old) needles and twigs. The collected samples were mixed evenly and placed into paper file bags. We selected roots with diameters greater than 2 mm for our research. The contents of N (%) and P (%) in the leaves were determined after sterilization at 105 • C, drying at 60 • C and mechanical grinding. The average value of each sample was taken to calculate the average contents of N (%) and P (%). Soil samples of the surface layer (0-20 cm) were collected under the selected tree. The soil samples were mixed fully and evenly. After air-drying in the laboratory, impurities were removed and the contents of N (g/kg) and P (g/kg) were determined after grinding and screening with 0.25 mm mesh. Roots of Pinus tabuliformis collection were carried out by a root-tracking method. The main roots of the sampled Pinus tabuliformis were found first, and the fine roots on the main roots were sequentially exposed by gradually removing sediment downward in the direction of the main roots. Roots containing at least five grades were cut off with pruning scissors, and these fine root samples were placed in selfsealing bags for preservation through temporary freezing. After taking the root samples back to the laboratory, the soil attached to the root samples was washed with water. The samples were graded according to the root order. The measured fine roots were placed in an oven at 60 • C for 72 h to maintain their weight. Root samples were kept for testing after crushing and screening with a 2 mm sieve. The total N content was determined by Kjeldahl determination, the leaf P content was determined by the molybdenum antimony anti-colorimetric method, and the soil total phosphorus was determined by the alkali fusion-Mo-Sb anti-spectrophotometric method [19][20][21]35]. The calculation method for the N mass and P mass of the leaves and roots of shrub species is the same as that for Pinus tabuliformis.

Data Analysis
Principal component analysis (PCA) was used to reduce the dimensions of the plant nutrient traits, root nutrient and soil nutrient factors and was conducted within the R environment using the "vegan" package.
Generalized additive models (GAMs) were used to evaluate the effects of soil nutrient factors on leaf and root nutrient traits. This approach utilizes both parametric and nonparametric components to reduce the model risks inherent to linear models [22]. The model can be summarized as: g(E(Yi)) = β 0 + S 1 (x i ) + S 2 (x i ) + e i (1) where g is a link function, E(Y i ) is the estimate for the responsible variable Y i , S 1 is the smooth function of x i for different light treatments, and S 2 is the smooth function of x i throughout the investigation time. x i (i = 1, 2, 3, . . . , 12) are the explanatory variables, and they are the number of new rhizomes, new rhizome length, new rhizome diameter, etc. β 0 is the constant term and e i is the error term. All calculations were conducted within the R environment using the "mgcv" package.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.