Changes in Plant Nitrogen Uptake Strategies Following Vegetation Recovery in Karst Regions

Abstract

Understanding plant nitrogen (N) uptake strategies during vegetation recovery is essential for restoring and rehabilitating degraded ecosystems. However, there are few studies on plant N uptake strategies in karst regions. In this study, space-for-time substitution was used to investigate the dynamic changes in plant N uptake strategies during vegetation restoration. Grassland, shrub–grassland, shrubland, and woodland naturally recovering in karst ecosystems were chosen as the research objects. The dominant species at each stage were investigated. Dominant plant N uptake rates were measured using the ¹⁵N labeling technique, and plant root functional traits and available soil N were determined. Our results showed that soil inorganic N content and composition varied significantly with vegetation recovery. In early vegetation recovery stages, the soil inorganic N content was low and dominated by ammonium (NH₄⁺), while in the late stages, soil inorganic N content increased, and nitrate (NO₃⁻) became the dominant form. In early vegetation recovery stages, dominant plants preferentially absorbed NH₄⁺, contributing to 90.3%–98.5% of the total N uptake. With vegetation recovery, plants increased the NO₃⁻ uptake ratio from 1.48%–9.42% to 30.1%–42.6%. Additionally, the root functional traits of dominant plants changed significantly during vegetation recovery. With vegetation recovery, specific root lengths and specific root areas decreased, while root N content and plant N uptake rates increased. In summary, plants developed N uptake strategies coordinated with soil N supply by modifying root functional traits following vegetation recovery in karst regions.

1. Introduction

Nitrogen (N) is a key nutrient that restricts plant growth in terrestrial ecosystems [1,2]. Besides small-molecule organic N, plants primarily utilize soil nitrate (NO₃⁻) and ammonium (NH₄⁺) [3,4,5]. Soil N availability affects plant growth and improvements in ecological function [6,7]. Approximately 57% of plant growth worldwide is limited in N content [8], which is attributed to lower overall soil N availability [6,9,10]. Moreover, if the soil-supplied N form does not match the plant’s preferred N form, plant growth may be limited, even if soil N availability is high [11,12,13]. Investigating plant N uptake strategies is the basis for an in-depth understanding of plant growth, community assembly, and succession [14,15,16], and it has become a hot topic in the current ecological plant restoration literature.

To better adapt to the environment, plants have developed different N uptake strategies, depending on the available soil N forms [17,18,19]. For example, Picea asperata and Abies faxoniana preferred NO₃⁻ uptake in China’s coniferous forest [20]. Larix kaempferi preferred NH₄⁺ uptake in Japan’s coniferous forest [21]. On the Tibetan Plateau, the N uptake preference of Picea abies changed from NH₄⁺ to NO₃⁻ with increasing forest age [22]. In addition, when the available soil N composition and content vary greatly, plants will adjust root functional traits to adapt to the soil environment. Research has shown that plants develop finer roots under low-N conditions [23], which improves the plant’s ability to absorb available N. Plants can also improve their N utilization capacity by extending their specific root length and area [24,25]. The ¹⁵N labeling technique can be used to quantify the plant’s ability to absorb different N sources (NH₄⁺, NO₃⁻, amino acids, etc.), thus clarifying plant N uptake preference [26]. Although studies on plant N uptake strategies advanced the understanding of N cycling, most have been conducted in non-karst areas.

Karst landscapes cover about 15% of the Earth’s surface [27,28,29] and are known for their rich species diversity and significant carbon sink potential [30,31,32]. Due to unique geological conditions and human disturbance, the karst ecosystem is delicate, with ecological and environmental issues, including biodiversity loss, severe soil erosion, and exposed rocks, resulting in large-scale “rocky desertification” [33,34,35]. Vegetation recovery is one of the most effective ways to control “rocky desertification”, but slow plant growth poses an issue to this process [6,27]. According to the literature, inadequate soil N supply is an important restriction of plant growth during early vegetation recovery phases in karst areas [36,37]. However, how plant N uptake strategies change during vegetation recovery is still unclear. Soils developed from carbonate rocks are characterized by high calcium content and pH in karst regions [7,27,38]. Calcium can be combined with soil organic matter, promoting its accumulation [39,40], while higher pH increases the activity of nitrifying microorganisms [41]. These factors affect soil N availability, potentially leading to the development of unique N uptake strategies in karst regions. Soil N availability is low in early vegetation recovery phases in karst regions [6]. In this habitat, surface vegetation is dominated by herbs and low shrubs, and plants may develop larger specific root lengths and areas to increase the capture of available N [25,42]. With vegetation recovery, shrubland and woodland are gradually forming, and litter input can increase soil organic matter accumulation, improving soil quality and environment, thereby leading to increased soil N availability [6,43]. Under higher pH background conditions, soil inorganic N content is high in later vegetation recovery stages in karst regions and may primarily present itself in NO₃⁻ [37]. Therefore, plants may develop different N uptake strategies to meet their growth requirements under conditions where soil inorganic N content and composition vary significantly in different vegetation recovery stages. We hypothesized that plants develop different N uptake strategies with vegetation recovery due to soil N availability and form changes in karst regions.

Space-for-time substitution was used to study plant N uptake strategies during vegetation recovery in karst regions for hypothesis verification. Grassland, shrub–grassland, shrubland, and woodland with natural recovery in karst regions were selected. Community composition and dominant species in different vegetation recovery stages were investigated using the survey plot method. This study aimed to clarify the changes in plant N uptake strategies and their driving factors during vegetation recovery by determining the plant root functional traits, available soil N content, and N uptake rates of dominant species in different vegetation recovery stages.

2. Materials and Methods

2.1. Site Description

The study area was located in Guilin, Guangxi Zhuang Autonomous Region, China (110°31′–110°54′ E, 25°12′–25°21′ N) (Figure 1), with an altitude of 259–381 m. The area experiences a mid-subtropical monsoon climate, with an average annual temperature of roughly 19.6 °C. There is abundant rainfall, with distinct wet and dry seasons: March to August is the rainy season, and September to the following February is the dry season. The average annual precipitation, sunshine time, and annual frost-free period are about 1712 mm, 1615 h, and 256–349 days, respectively. The region has a typical peak-cluster depression landform, and the IUSS Working Group WRB has designated the studied soil as calcareous Alfisol. Additionally, humans have cut down large areas of natural vegetation and planted crops on steep slopes and in rock crevices throughout time. The regions have undergone long periods of agricultural cultivation, resulting in significant soil erosion and rocky desertification. Over time, areas where rocky desertification became too severe for farming were gradually abandoned. After decades of natural recovery, different vegetation recovery stages have emerged.

Four typical recovery stages, i.e., grassland, shrub–grassland, shrubland, and woodland, were selected for study based on the field survey results (

Table 1. Fundamental details of the study sites at different recovery stages.

Recovery Stages	Longitude and Latitude	Altitude (m)	Slope (°)	Soil Depth (cm)	Interference Conditions
Grassland	110°32′ E 25°12′ N	324–341	2–5	0–30	Abandoned, no interference for 3–5 years
Shrub–grassland	110°32′ E 25°12′ N	322–348	4–8	0–30	Abandoned, no interference for 7–10 years
Shrubland	110°53′ E 25°21′ N	321–343	12–15	0–25	Abandoned, no interference for 30–35 years
Woodland	110°52′ E 25°20′ N	246–300	20–25	0-20	Abandoned, no interference for 70–80 years

). Grassland vegetation was relatively homogeneous. In the shrub–grassland stage, the presence of herbaceous plant species increased significantly, while some lianas and low shrubs appeared, forming a diverse vegetation community. In the shrubland stage, shrubs became the dominant layer, with a significant increase in vegetation depression. In the woodland stage, tall trees became the dominant vegetation layer, forming an obvious canopy layer. Under the tree layer, there were also shrubs and herbs, and the overall surface vegetation coverage was higher, with a more complex and diversified vegetation structure. Four representative sample plots were set up in each recovery stage for the vegetation survey, with spacing between sample plots greater than 300 m. The results showed the following dominant species: Miscanthus floridulus in the grassland; Bauhinia championii, Alchornea trewioides, Miscanthus floridulus, and Selaginella moellendorffii in the shrub–grassland; Loropetalum chinense, Bauhinia championii, Carex capilliformis, and Paederia foetida in the shrubland; and Cyclobalanopsis glauca, Mallotus philippinensis, Murraya paniculata, Trachelospermum jasminoides, and Carex sendaica in the woodland.

2.2. Field Hydroponic Experimental Design

According to the importance values of the plant community diversity survey, 1–3 dominant plants were selected as the study species in each recovery stage, listed as follows: Miscanthus floridulus in the grassland; Bauhinia championii, Alchornea trewioides, and Miscanthus floridulus in the shrub–grassland; Loropetalum chinense and Bauhinia championii in the shrubland; and Cyclobalanopsis glauca and Mallotus philippinensis in the woodland. There were 5 replicates for each species, so a total of 40 plants needed to be marked.

This study concentrated on the plant uptake of NH₄⁺, NO₃⁻, and organic N, with glycine used as a model marker for organic N [44]. Three ¹⁵N-labeled solutions (¹⁵NH₄NO₃ + C₂H₅NO₂, NH₄¹⁵NO₃ + C₂H₅NO₂, and NH₄NO₃ + C₂H₅¹⁵NO₂) and one unlabeled solution (NH₄NO₃ + C₂H₅NO₂) were prepared. The ¹⁵N atom% of ¹⁵NH₄NO₃ and NH₄¹⁵NO₃ was 10%, while the ¹⁵N atom% of C₂H₅¹⁵NO₂ was 99%. The N contents of NH₄⁺, NO₃⁻, and glycine in each treatment were all 150 μmol N L⁻¹. Additionally, 10 mg L⁻¹ ampicillin was added to each treatment to reduce microbial activity and prevent amino acid decomposition, along with 0.2 mmol CaCl₂ to preserve root function and integrity [45]. The prepared solutions were transferred into 15 mL centrifuge tubes and subsequently labeled.

Five similar-sized plants were chosen randomly for each species. Along the stem base, fine roots under 2 mm in diameter were dug out diagonally in four directions. After gently cleaning with deionized water, the roots were submerged in the appropriate labeling solutions. The length of the labeled fine root was approximately 15 cm. Then, the time that plant roots were placed in the solution was accurately recorded. After 2 h of labeling, the roots were cut at the location of the labeling solution. The fine roots were then carefully washed with deionized water after submerging in the KCl solution (50 mmol L⁻¹) for 3 min. Finally, the fine roots were wiped with clean laboratory paper and then placed in a labeled envelope. After being brought back to the laboratory, the fine roots were stored in a 4 °C refrigerator.

2.3. Plant Root Functional Traits

A root scanner was used to scan the roots, and the data were analyzed with WinRHIZO to obtain root surface area, volume, and length. The scanned roots were returned to their original envelopes and dried to a consistent weight for 48 h at 70 °C in an oven. Dry root weight was determined by the millionth balance. Fine roots were ground to powder with an AM410 Planetary Ball Mill. Root N content (RN) and ¹⁵N atom% of fine root samples were determined using a Sercon Integra 2 Elemental Analyzer (Sercon Ltd., Crewe, UK).

The formulas for specific root length (SRL), specific root area (SRA), and root length density (RLD) were calculated as follows:

$$\mathrm{SRL}\left({\mathrm{m} \mathrm{g}}^{-1}\right)={\displaystyle \frac{\mathrm{L}}{\mathrm{M} \times 100}}$$

(1)

$$\mathrm{SRA}\left({{\mathrm{cm}}^2 \mathrm{g}}^{-1}\right)={\displaystyle \frac{\mathrm{S}}{ \mathrm{M}}}$$

(2)

$$\mathrm{RLD}\left({\mathrm{cm} \mathrm{cm}}^{-3}\right)={\displaystyle \frac{\mathrm{L}}{\mathrm{V}}}$$

(3)

where L, M, S, and V represent root length (cm), dry root weight (g), root surface area (cm²), and root volume (cm³), respectively.

2.4. Plant Nitrogen Uptake Strategies

The plant N uptake rate was calculated by Formula (4) [45]:

$$\mathrm{NUR}={\displaystyle \frac{{\mathrm{N}}_{\mathrm{Root}} \times {\mathrm{APE}}_{\mathrm{sample}}}{\mathrm{T} \times {\mathrm{APE}}_{\mathrm{added}}}}$$

(4)

where NUR (μg N g⁻¹ h⁻¹), N_root (μg g⁻¹), T, APE_sample, and APE_added represent the plant N uptake rate, root N content, labeling time (h), the difference in atom% ¹⁵N between ¹⁵N-labeled and unlabeled roots, and atom% ¹⁵N of the added markers, respectively.

Considering that there were differences between the actual soil N contents and the experimentally added N contents, the ratio of different N form uptake to total N uptake was corrected according to the actual contents of soil NH₄⁺, NO₃⁻, and glycine. Taking NH₄⁺ as an example, we can determine the following formula:

$${\beta }_{{NH}_4^{+}}=(\mathrm{A}\mathrm{M}\mathrm{U}\times {\mathrm{C}}_{{\mathrm{N}\mathrm{H}}_4^{+}})/(\mathrm{A}\mathrm{M}\mathrm{U}\times {\mathrm{C}}_{{\mathrm{N}\mathrm{H}}_4^{+}}+\mathrm{N}\mathrm{T}\mathrm{U}\times {\mathrm{C}}_{{\mathrm{N}\mathrm{O}}_3^{-}}+\mathrm{G}\mathrm{L}\mathrm{Y}\mathrm{U}\times {\mathrm{C}}_{\mathrm{G}\mathrm{L}\mathrm{Y}})$$

(5)

where β_NH4⁺, AMU (μg N g⁻¹ h⁻¹), C_NH4⁺ (μg g⁻¹), NTU (μg N g⁻¹ h⁻¹), C_NO3⁻ (μg g⁻¹), GLYU (μg N g⁻¹ h⁻¹), and C_Gly (μg g⁻¹) represent the NH₄⁺-uptake-to-total-N-uptake ratio, NH₄⁺ uptake rate by plants, soil NH₄⁺ content, NO₃⁻ uptake rate by plants, soil NO₃⁻ content, glycine uptake rate by plants, and soil glycine content, respectively.

2.5. Available Soil Nitrogen

Soils were collected from 0 to 15 cm near the roots of each plant in all four directions and mixed into one sample, yielding a total of forty soil samples. A 2 mm sieve was used to filter soil samples following the removal of the stones, litter, and roots. Then, 50 mL of 0.05 mol L⁻¹ K₂SO₄ was used to extract fresh soil equal to 10 g of dry soil weight. The extraction solutions were stored in a refrigerator at 4 °C, and the contents of NH₄⁺ and NO₃⁻ were determined using a flow analyzer (Skalar, Breda, The Netherlands) [6], while the free amino acid contents were measured via high-performance liquid chromatography(HPLC-MS/MS API3200 Q-TRAP, Foster City, CA, USA) [45].

2.6. Data Statistics and Analysis

The variations in plant N uptake rates, root functional traits, and available soil N contents in different vegetation recovery stages were compared using one-way analysis of variance (ANOVA) and Tukey’s test in SPSS 24.0 (IBM, Chicago, IL, USA). In Origin 2021 (OriginLab Corp., Northampton, MA, USA), a correlation plot was used to examine the relationship between plant N uptake rates and ratios with root functional traits and available soil N contents. The impact of available soil N contents and plant root functional traits on plant N uptake was examined using structural equation modeling (Amos 28.0, IBM, Chicago, IL, USA).

3. Results

3.1. Soil Nitrogen Availability

Available soil N content and composition varied considerably with vegetation recovery (

Table 2. Inorganic and organic nitrogen contents (mean ± SD) in the soil of dominant plants (n = 5) in different vegetation recovery stages.

Dominant Plants	Recovery Stages	NH4+ (mg kg−1)	NO3− (mg kg−1)	Glycine (mg kg−1)	TFAA (mg kg−1) A	Inorganic N (mg kg−1)	NH4+/NO3− ratio
Miscanthus floridulus	Grassland	8.51 ± 5.72 de	5.92 ± 2.88 d	0.03 ± 0.01 b	0.59 ± 0.12 ab	14.4 ± 7.62 de	1.52 ± 1.04 bc
Miscanthus floridulus	Shrub–grassland	5.83 ± 1.00 e	5.71 ± 3.05 d	0.06 ± 0.02 a	0.81 ± 0.10 a	11.5 ± 3.47 e	1.31 ± 0.72 cd
Alchornea trewioides		29.3 ± 4.43 a	13.3 ± 2.92 d	0.04 ± 0.02 ab	0.56 ± 0.39 abc	42.6 ± 3.94 ab	2.32 ± 0.85 ba
Bauhiniachampionii		19.8 ± 3.91 b	6.20 ± 2.70 d	0.04 ± 0.01 ab	0.44 ± 0.09 bc	26.0 ± 6.05 cd	3.62 ± 1.24 a
Loropetalum chinense	Shrubland	8.84 ± 4.12 de	34.6 ± 5.27 ab	0.03 ± 0.01 b	0.27 ± 0.11 c	43.4 ± 9.35 ab	0.24 ± 0.09 e
Bauhinia championii		12.7 ± 5.09 cd	26.0 ± 12.1 bc	0.03 ± 0.01 b	0.27 ± 0.26 c	38.8 ± 16.7 bc	0.52 ± 0.16 de
Cyclobalanopsis glauca	Woodland	15.3 ± 3.44 bc	39.2 ± 13.1 a	0.03 ± 0.00 b	0.31 ± 0.12 bc	54.6 ± 15.4 a	0.43 ± 0.17 de
Mallotus philippinensis		15.0 ± 4.40 bc	24.0 ± 8.54 c	0.04 ± 0.02 ab	0.40 ± 0.20 bc	39.0 ± 8.79 bc	0.70 ± 0.38 ced

^A TFAA, total free amino acid. At the p < 0.05 level, significant changes from different dominant plants are represented by different lowercase letters. Notes: If the mean is not specified in the results, this means that the mean value for each dominant species was analyzed. If the mean is specified, it refers to the overall mean of all of the dominant species in each recovery stage.

). Compared with the grassland and shrub–grassland (14.4–26.7 mg kg⁻¹), the average soil inorganic N contents in the shrubland and woodland were significantly higher, ranging from 41.1 to 46.8 mg kg⁻¹. Soil inorganic N was mainly NH₄⁺ (5.83–29.3 mg kg⁻¹) in the grassland and shrub–grassland but dominated by NO₃⁻ (26.0–39.2 mg kg⁻¹) in the shrubland and woodland. The soil NH₄⁺/NO₃⁻ ratios were greater than one in the grassland and shrub–grassland and less than one in the shrubland and woodland. The total free amino acid and glycine contents in soil were very low in all recovery stages, ranging from 0.27 to 0.81 mg kg⁻¹ and 0.03 to 0.06 mg kg⁻¹, respectively, which were considerably lower than the soil inorganic N content. The soil NH₄⁺ content of Bauhinia championii in the shrub–grassland (19.8 mg kg⁻¹) was remarkably higher than that in the shrubland (12.7 mg kg⁻¹), whereas the soil NO₃⁻ content of Bauhinia championii in the shrub–grassland (6.20 mg kg⁻¹) was significantly lower than that in the shrubland (26.0 mg kg⁻¹).

3.2. Root Functional Traits of Dominant Plants

The root functional traits of dominant plants varied significantly during vegetation recovery (Figure 2). Compared to grassland and shrub–grassland (17.2–18.5 m g⁻¹, 197–253 cm² g⁻¹, and 4.48–9.16 g kg⁻¹, respectively), the average specific root length (SRL) and specific root area (SRA) of dominant plants in the shrubland and woodland decreased to 9.07–11.7 m g⁻¹ and 127–144 cm² g⁻¹, respectively, whereas the average root N content (RN) of dominant plants increased to 12.1–16.1 g kg⁻¹. Except for Miscanthus floridulus in the shrub–grassland, there were no appreciable variations in the dominating plants’ root length density (RLD) during vegetation recovery. With vegetation recovery, the SRL and RLD of Miscanthus floridulus and the RN of Bauhinia championii significantly increased.

3.3. Nitrogen Uptake Rate and Ratio of Dominant Plants

During vegetation recovery, the N uptake rates of dominant plants varied considerably (Figure 3). Except for Cyclobalanopsis glauca, the NH₄⁺ uptake rates (AMU) of dominant plants in the shrubland and woodland increased from 8.86–16.3 μg N g⁻¹ h⁻¹ to 19.6–29.3 μg N g⁻¹ h⁻¹ compared with those in the grassland and shrub–grassland. Compared to the grassland and shrub–grassland, the N uptake rates of dominant plants in the shrubland and woodland exhibited a significant increase: NO₃⁻ uptake rates (NTU) increased from 0.62–1.13 μg N g⁻¹ h⁻¹ to 3.38–8.19 μg N g⁻¹ h⁻¹; glycine uptake rates (GLYU) increased from 2.75–5.12 μg N g⁻¹ h⁻¹ to 5.19–8.32 μg N g⁻¹ h⁻¹; and total N uptake rates (TNU) increased from 12.8–22.6 μg N g⁻¹ h⁻¹ to 26.3–42.7 μg N g⁻¹ h⁻¹. The NH₄⁺ uptake rate of dominant plants was higher than NTU and GLYU. The NO₃⁻ uptake rate, GLYU, and TNU of Bauhinia championii significantly increased with vegetation recovery.

The ratios of different N form uptake to total N uptake by dominant plants changed significantly during vegetation recovery (Figure 4). In the grassland and shrub–grassland, the NH₄⁺ uptake ratios (β_NH4⁺) of dominant plants ranged from 90.3% to 98.5%, whereas the NO₃⁻ uptake ratios (β_NO3⁻) were only 1.48%–9.42%. In the shrubland and woodland, β_NH4⁺decreased to 57.4%–69.8%, while β_NO3⁻ increased to 30.1%–42.6%. The ratios of glycine uptake (β_GLY) in dominant plants remained low, ranging from 0.03% to 0.32% during vegetation recovery. With vegetation recovery, the β_NH4⁺ of Miscanthus floridulus and Bauhinia championii decreased, while β_NO3⁻ increased.

3.4. Drivers of the Nitrogen Uptake Rate and Ratio of Dominant Plants

The NH₄⁺ uptake rate, NTU, and TNU showed strong and positive correlations with the NH₄⁺ content and RN, and significant negative correlations with the NH₄⁺/NO₃⁻ ratio (Figure 5). The NO₃⁻ uptake rate and TNU were also significantly and negatively correlated with SRL. The NH₄⁺ uptake ratio showed a significant positive correlation with glycine content, as well as the SRL, SRA, and NH₄⁺/NO₃⁻ ratio, and significant negative correlations with the NO₃⁻ content and RN, whereas β_NO3⁻ correlated with the opposite of these indicators. According to the structural equation model analysis, soil inorganic N and NTU directly affected β_NO3⁻, while plant root functional traits mainly indirectly affected β_NO3⁻ by regulating plant NTU (Figure 6).

4. Discussion

4.1. Plant Nitrogen Uptake Strategies During Vegetation Recovery

The uptake rates of NH₄⁺ (AMU), NO₃⁻ (NTU), and glycine (GLYU) in dominant plants in karst regions were 8.86–29.3 μg N g⁻¹ h⁻¹, 0.62–9.18 μg N g⁻¹ h⁻¹, and 2.75–8.32 μg N g⁻¹ h⁻¹, respectively (Figure 3), which were higher than those reported in cold–temperate, temperate, and tropical forests [21,45]. Liu et al. [45] found that plant AMU, NTU, and GLYU ranged from 6.77 to 12.8 μg N g⁻¹ h⁻¹, 0.13 to 2.17 μg N g⁻¹ h⁻¹, and 0.88 to 5.93 μg N g⁻¹ h⁻¹ in tropical and temperate forests, respectively. Additionally, all dominant plants showed significantly higher AMU than NTU and GLYU (Figure 3), and the results were consistent with previous studies in cool–temperate, temperate, subtropical, and tropical forests [21,45,46]. The uptake rates of NH₄⁺, NO₃⁻, and glycine in Bauhinia championii significantly increased with vegetation recovery. We also found that compared to the grassland and shrub–grassland (12.8–22.6 μg N g⁻¹ h⁻¹), the TNU of dominant plants significantly increased in the shrubland and woodland (26.3–42.7 μg N g⁻¹ h⁻¹), indicating that dominant plants improved N uptake efficiency during vegetation recovery (Figure 3). Validating the findings of Yi et al. [16], plants in the late recovery stage would acquire available soil N by increasing the physiological uptake efficiency (plant N uptake rate). The glycine-to-total-N-uptake ratio of dominant plants was only 0.03%–0.32%, which was nearly negligible, indicating that dominant plants primarily absorbed soil inorganic N. Li et al. [47] found similar results that the utilization of organic acids by plants was low under high inorganic N levels. With vegetation recovery, the NH₄⁺-to-total-N-uptake ratio (β_NH4⁺) of dominant plants decreased significantly, while the NO₃⁻-to-total-N-uptake ratio (β_NO3⁻) increased significantly (Figure 4). The opposite was noted in subtropical forests: plants increased β_NH4⁺ and decreased β_NO3⁻ with succession [16]. This result suggested that the changes in plant N uptake strategies in karst regions were indeed different from those in non-karst regions with vegetation recovery and helped to enhance the understanding of N cycling in karst regions.

Changes in plant N uptake strategies during vegetation recovery in karst regions might be related to available soil N supply. Studies found that plant N uptake preferences not only had direct positive feedback effects with soil mineralization and nitrification rates [48], but were also closely related to available soil N [49,50,51]. In this study, soil inorganic N content and dominant forms changed significantly with vegetation recovery (Table 2), which might be related to changes in soil N transformation processes. Previous studies had found that soil mineralization and nitrification rates increased significantly with recovery in karst regions [6,7,52]. This might be because soils are usually clay-heavy and have low organic matter content in karst regions in early vegetation recovery phases [6,7]. These conditions hinder soil microbial growth, which may inhibit soil mineralization and nitrification processes [6,7]. In our study, soil inorganic N was low and mainly NH₄⁺ in the grassland and shrub–grassland (Table 2), and plants generally preferred to absorb the dominant N in soil [46,53], resulting in higher β_NH4 in dominant plants (Figure 4). Additionally, plants require lower energy consumption to absorb NH₄⁺ than NO₃⁻ [20,45], which might also be an important factor for the higher β_NH4⁺ in plants. In later vegetation recovery stages, soil organic matter accumulation improved soil quality and environment, which significantly increased soil mineralization rates [40,41]. Combined with the high pH in karst regions, NH₄⁺ from mineralization could be easily converted to NO₃⁻ through nitrification [54,55]. In our study, soil inorganic N was high and dominated by NO₃⁻ in the shrubland and woodland (Table 2), and β_NO3⁻ in dominant plants increased significantly to obtain more available N (Figure 4). The correlation analysis found that β_NH4⁺ had a strong negative correlation with the NH₄⁺/NO₃⁻ ratio and a significant positive correlation with NO₃⁻ content, while β_NO3⁻ was the opposite (Figure 5), further confirming that the dominant form of soil inorganic N could significantly affect plant N uptake strategies. The research results could provide a reference basis for optimizing the NH₄⁺/NO₃⁻ ratio to improve the efficiency of N fertilizer use by regulating the rate of the N transformation process in agricultural management in karst regions.

4.2. Relationship Between Plant Root Functional Traits and Plant Nitrogen Uptake Strategies During Vegetation Recovery

Plants regulate their root traits to obtain N sources for better environmental adaptation [46,56,57]. In this study, β_NH4⁺ in dominant plants was significantly positively correlated with specific root length (SRL) and specific root area (SRA) and significantly negatively correlated with root nitrogen (RN) content, whereas β_NO3⁻ in dominant plants was exactly the opposite (Figure 5). These results revealed the important role played by plant root functional traits in N uptake. Previous studies have demonstrated a synergistic link between plant roots’ capacity for exploration and nutrient uptake [16,58]. The available soil N content was low in the grassland and shrub–grassland. To adapt to this environment, plants developed larger SRL and SRA to efficiently obtain resources [59,60]. However, with vegetation recovery, the amount of inorganic N in the soil increased significantly, along with a corresponding increase in plant RN content in the shrubland and woodland. Under these circumstances, plants would explore less soil space and allocate more resources to improve their defenses by developing thicker roots (e.g., decreasing SRL and SRA and increasing tissue density) [61,62,63]. Additionally, plants increased the physiological root uptake rate to correspond to the higher nutrient demand [16]. Dominant plants had smaller SRL and SRA but higher NTU and TNU in the shrubland and woodland than in the grassland and shrub–grassland. These results suggested that plants preferentially acquired N by increasing uptake efficiency in N-rich environments. Notably, NO₃⁻ was more mobile in soil and could more easily reach the roots than NH₄⁺ [20,64]. In order to obtain more available N from the soil, dominant plants in the shrubland and woodland increased β_NO3⁻, which has a higher content and greater mobility in the soil. Furthermore, the structural equation model showed that soil inorganic N, plant N uptake rate, and plant root functional traits could significantly affect β_NO3⁻ (Figure 6). In summary, the study results verified our hypothesis that when soil inorganic N content and composition changed during vegetation recovery, plants would increase β_NO3⁻ by altering characteristics, including SRL, SRA, RN, and plant N uptake rate, to adapt to the environment. The response of plant N uptake strategies to plant root functional traits could provide a reference basis for species selection for vegetation restoration in degraded karst ecosystems.

5. Conclusions

With vegetation recovery, the soil inorganic N content significantly increased and changed from NH₄⁺-dominated to NO₃⁻-dominated. Plants in late recovery stages had relatively lower SRL and SRA and higher RN than those in early recovery stages (when plants primarily uptake NH₄⁺), which improved NO₃⁻ uptake and changed N uptake strategies. Ultimately, this study’s findings enhance our understanding of karst ecosystems’ N cycle and provide a theoretical foundation for agricultural N management and species selection in degraded ecosystems.